Purified preparations of recombinant human interferon-gamma (rIFN-gamma) with Cys-Tyr-Cys at the N-terminus ([ Cys-Tyr-Cys]IFN-gamma) derived from Escherichia coli gave two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two peaks on reversed-phase high-performance liquid chromatography (rpHPLC). In contrast, rIFN-gamma without Cys-Tyr-Cys and rIFN-gamma in which both Cys-1 and Cys-3 were substituted with serine behaved as a single species on both SDS-PAGE and rpHPLC. These results suggest that the N-terminal portion of rIFN-gamma is heterogeneous. To elucidate the structure of the N-terminal portion, the N-terminal peptide preparation was obtained by binding rIFN-gamma to thiopropyl-Sepharose 6B gel with disulfide linkage followed by trypsin digestion and elution with 2-mercaptoethanol. The preparation gave four peaks (NT-1, NT-2, NT-3, and NT-4, in order of elution) on rpHPLC; all four were found to be Cys-1-Lys-9 by amino acid analysis after acid hydrolysis. Various analyses indicate that NT-1 is the intact nonapeptide, that NT-3 and NT-4 are N alpha-formyl and N alpha-acetyl forms of NT-1, respectively, and that NT-2 may be S-blocked at Cys-1. It is concluded that E. coli-derived [Cys-Tyr-Cys]IFN-gamma is partially N alpha-acylated. The data also suggest that N alpha-acylation does not affect the biological activity of [Cys-Tyr-Cys]IFN-gamma.