The insulin-like growth factor I gene is transcribed from two promoter regions, resulting in alternative first exons in insulin-like growth factor I messenger RNAs. A previous study showed that the sequence from− 73 to +44 (where +1 is the first nucleotide in the exon 2 transcription initiation cluster) contained an active exon 2 promoter, and that sequences between −73 and −36 were required for promoter activity. In the current study, the roles of two putative cis-acting elements within the −73 to +44 region in basal exon 2 promoter activity were evaluated using mutagenesis and nuclear protein-DNA binding assays. Mutation of the CCCCACCC sequence at position −53 to GAAATCCC resulted in a complete loss of promoter activity in transient transfection assays in GH₃, OVCAR-3, C6, and Chinese hamster ovary (CHO) cells. A −73/+24 exon 2 promoter-luciferase construct had partial promoter activity. Mutation of a putative initiator motif surrounding the major exon 2 start site did not alter the activity of this construct. In electrophoretic mobility shift assays, a ³²P-labeled oligomer extending from −73 to +44 in the exon 2 promoter was specifically bound by GH₃ cell nuclear extracts. A ³²P-labeled oligomer which extended from −63 to −37 in the exon 2 promoter was specifically bound by GH₃ and OVCAR-3 cell nuclear extracts. These unlabeled oligomers inhibited the binding of a labeled− 236/+44 exon 2 promoter fragment to OVCAR-3 nuclear extracts. Mutation of the CCCCACCC sequence prevented the unlabeled −73/+44 oligomer from inhibiting the binding of the −236/+44 fragment. An unlabeled oligomer containing a consensus activating protein-2 (AP-2)-binding site inhibited labeled −236/+44, −73/+44, and− 63/−37 exon 2 promoter binding with a much lower affinity than did the respective unlabeled oligomers. Purified AP-2 protein did not bind to the exon 2 promoter fragment, nor did anti-AP-2 antibody alter the binding. Cotransfection of AP-2 expression vector did not significantly increase exon 2 promoter activity. On the other hand, an oligomer containing a consensus Sp1-binding site inhibited labeled −63/−37 exon 2 promoter binding by GH₃ cell nuclear extracts with an affinity similar to that of the unlabeled −63/−37 oligomer. A mutation in the Sp1-binding site in this same oligomer resulted in a complete loss of binding affinity. Purified Sp1 bound to the −63/−37 exon 2 promoter oligomer. Addition of polyclonal antibody to Sp1 resulted in a partial supershift of the complex formed between GH₃ cell and OVCAR-3 cell nuclear extracts and the labeled− 63/−37 oligomer. However, in Drosophila Schneider cells, which are an experimental model for studying the ability of Sp1 to activate transcription, the −73/+44 exon 2 promoter construct was not stimulated by cotransfection with an Sp1 expression plasmid. UV cross-linking studies indicated that proteins of approximate molecular mass 125, 76, 47, and 38 kDa are bound to the proximal (−236/+44) exon 2 promoter region. It is concluded that the CCCCACCC sequence at −53 is required for exon 2 promoter activity. Moreover, specific binding of nuclear proteins to the proximal exon 2 promoter region requires the CCCCACCC sequence. Sequences downstream of the exon 2 initiation site from +24 to +44 are required for full promoter activity. However, the putative initiator surrounding the major transcription start site at +1 does not appear to be important for the strength of the proximal promoter. The CCCCACCC sequence at −53 appears to be a CACCC box, which binds zinc finger transcription factors of the Kruppel family such as Sp1, although protein factors in addition to Sp1 are required to activate exon 2 …