The human cytomegalovirus IRS1 and TRS1 open reading frames encode immediate-early proteins with identical N-terminal domains and divergent C-terminal regions. Both proteins have been shown previously to activate reporter genes in transfection assays in cooperation with other viral gene products. We have constructed two viruses carrying substitution mutations within either the IRS1 or TRS1 open reading frame. ADsubIRS1 failed to produce the related IRS1 and IRS1(263) proteins, but it replicated with normal kinetics to produce a wild-type yield in human fibroblasts. The addition in trans of the IRS1(263) protein, which antagonizes the ability of IRS1 and TRS1 proteins to activate reporter genes, did not inhibit the growth of the mutant virus. ADsubTRS1 failed to produce the TRS1 protein, and it generated an approximately 200-fold-reduced yield of infectious virus in comparison to its wild-type parent. Viral DNA accumulated normally, as did a set of viral mRNAs that were monitored in ADsubTRS1-infected cells. However, two tegument proteins were partially mislocalized and infectious virus particles did not accumulate to normal levels within ADsubTRS1-infected cells. Further, infectious ADsubTRS1 particles sedimented abnormally in a glycerol-tartrate gradient, indicating that the structure of the mutant particles is aberrant. Our analysis of the ADsubTRS1 phenotype indicates that the TRS1 protein is required, either directly or indirectly, for efficient assembly of virus particles.