Specific missense mutations of the vaccinia virus D13L gene confer resistance to the effects of rifampicin on virion morphogenesis. We constructed a recombinant vaccinia virus in which elements of the Escherichia coli lac operator system were used to regulate the D13L gene. Replication of the recombinant vaccinia virus was dependent on addition of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the virus yield was decreased by more than 99% when IPTG was omitted. Under the nonpermissive condition, transcription of the D13L gene was reduced and synthesis of the 65,000-Da protein product was inhibited by more than 95%. Consequently, virion morphogenesis was blocked at an early stage and uncoated membrane precursors of the immature viral envelope and uncleaved precursors of the major core proteins accumulated. The phenotype of the conditional lethal mutant virus, in the absence of IPTG, closely resembled that of wild-type virus in cells treated with rifampicin.