We have utilized insertional mutagenesis to investigate the functional importance of murine cytomegalovirus (MCMV) immediate early (IE or alpha) region 2 (ie2) in replication. We constructed a recombinant virus, RM408, that carries the Escherichia coli lacZ gene under transcriptional control of the MCMV alpha promoter/enhancer inserted within the ie2 transcription unit. RM408 carries the first defined genetic mutation in a specific CMV gene. After infection of NIH3T3 cells, RM408 expressed beta-galactosidase in abundance and with kinetics indistinguishable from those of the natural ie1 gene product which is left intact in the recombinant. We detected no expression from the ie2 region in RM408-infected cells. Growth kinetics, yield, and expression of other viral genes in RM408 was similar to wild-type MCMV, indicating that the ie2 gene product was nonessential for growth in cell culture.