A vaccinia virus host range mutant (hr mutant) deleted of 18 kilobase pairs at the left end of the genome has been employed to precisely map a viral gene required for multiplication in human cells. DNA fragments from the wild-type virus were inserted into the thymidine kinase gene of mutant virus by means of in vivo homologous recombination, and the recombinants obtained were screened for their ability to multiply in human cells. A short sequence, 855 base pairs long, overlapping the HindIII M and K fragments was able to restore a normal host range on the mutant virus. A single long open reading frame that could encode a polypeptide of 32.5 kDa was found in the nucleotide sequence of the host range gene. The direction of transcription and the length of the open reading frame are in excellent agreement with previous mapping of mRNAs within this region of the genome. In vitro translation of infected cell early mRNA, selected by hybridization to the host range gene, yielded a prominent polypeptide product whose size (29 kDa) was close to that expected from the predicted amino acid sequence. The phenotype of the hr mutant suggests that the host range gene plays a role in maintaining a high level of protein synthesis in human cells. It may behave positively by complementing the lack of an analogous cellular activity or negatively by antagonizing a cell function that inhibits viral multiplication.