The significance of the C-terminal part of human interferon gamma (hIFNγ) for its biological activity was studied by 3^′-end gene mutagenesis. A series of nine derivative genes obtained by systemic deletion of three codons was constructed and expressed in Escherichia coli LE392. It was shown that the yield of recombinant protein gradually decreased and the solubility gradually increased with truncation of the C terminus. To avoid artifacts related to the imperfect folding of the proteins during purification, the biological activity of the hIFNγ proteins was measured in clear cell lysates containing the soluble fractions only. The deletion of the C terminus had a two-step effect on both hIFNγ antiviral and antiproliferative activities. Whereas the removal of the last 3, 6, and 9 C-terminal amino acids led to a gradual increase (up to 10 times) in biological activity of hIFNγ, the deletion of more than 9 amino acids had an opposite effect. The truncation of the whole unstructured C-terminal domain resulted in a 10-fold decrease (but not in a complete loss) in biological activity of hIFNγ. The latter was sequestered upon deletion of 24 amino acids, 3 of which belonged to the α-helical domain F.