We examined the synthesis and processing of the polymerase polyprotein in RNA(-) temperature sensitive mutant of murine coronavirus strain A59. These temperature sensitive mutants of MHV-A59 synthesize viral RNA at the permissive temperature (33.0 degrees C), but are unable to synthesize viral RNA at the nonpermissive temperature (39.5 degrees C). The ts mutants have been mapped to five different complementation groups in the polymerase gene. The 5'-most complementation groups, Groups A and B, map to a region encoding an autoproteinase responsible for the cleavage of p28, the amino-terminal product of the polymerase polyprotein. We screened six temperature sensitive mutants to determine if there was an alteration in the proteolytic processing of the polymerase polyprotein, particularly in the cleavage of the p28 protein. Two mutants, tsNC9 and tsLA16, had altered proteolytic products at both the permissive and nonpermissive temperatures. One Group B temperature sensitive mutant, designated tsNC11, was defective in the production of p28 protein at the nonpermissive temperature. To further localize the site of the mutation in tsNC11, RNA representing the 5'-most 5.3 kb region of the polymerase gene was transfected into tsNC11-infected cells and virus production monitored. The transfected RNA was able to complement the defect in tsNC11, resulting in viral RNA synthesis and production of viral particles at the nonpermissive temperature. These results indicate that a gene product from the 5.3 kb region of gene 1 is required for coronavirus RNA synthesis.