Hepatoma-derived growth factor (HDGF) is an acidic polypeptide with mitogenic activity for fibroblasts performed outside the cells despite the presence of a putative nuclear localization signal (NLS). We have now cloned three related mouse cDNAs: one for a mouse homologue of human HDGF and two for additional HDGF-related proteins provisionally designated HDGF-related proteins 1 and 2 (HRP-1 and -2). Their deduced sequences have revealed that HDGF belongs to a new gene family with a highly conserved 98-amino-acid sequence at the amino terminus (hathregion, forh omologous to thea minot erminus ofH DGF). HRP-1 and HRP-2 proteins are 46 and 432 amino acids longer than mouse HDGF, respectively, and have no conserved amino acid sequence other than thehathregion. HRP-1 is a highly acidic protein (26% acidic) and also has a putative NLS. HRP-2 protein carries a mixed charge cluster, a sharp switch of positive- to negative-charge residues, which is often found in some nuclear proteins. Northern blotting shows that mouse HDGF and HRP-2 are expressed predominantly in testis and skeletal muscle, to intermediate extents in heart, brain, lung, liver, and kidney, and to a minimal extent in spleen. HRP-1 is expressed specifically in testis. These findings suggest that the HDGF gene family might play a new role in the nucleus especially in testis.