The 5' most gene of the murine coronavirus genome, gene 1, is presumed to encode the viral RNA-dependent RNA polymerase. cDNA clones representing this gene encompass more than 22 kilobases, suggesting that this region may encode multifunctional polyprotein(s). It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To identify possible functional domains of gene 1 and further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of the murine coronavirus mouse hepatitis virus strain JHM were subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. Although p28 is encoded from the first 1 kilobase at the 5'-end of the genome, translation of in vitro transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3 kilobase region was synthesized. This result suggests that the region close to 5.3 kilobases from the 5'-end of the genomic RNA is essential for the proteolytic cleavage and may contain an autoproteolytic activity. Addition of the protease inhibitor ZnCl2 blocked cleavage of the p28 protein. Site-directed mutagenesis of Cys residue 1137 significantly reduced the cleavage of the p28 protein, indicating that this residue, probably in conjunction with a downstream domain, plays an essential role in the cleavage of p28. This Cys residue may be part of a papain-like autoprotease encoded by gene 1.