Very little is known about the human cytomegalovirus functions that activate gamma (late) gene expression. We have investigated the regulation of the human cytomegalovirus gamma gene encoding the ICP36 major late DNA-binding protein family (UL44). Transactivation of the ICP36 gene promoter was found to be absolutely dependent on the trs1 gene product when expressed in cells in conjunction with ie1 and ie2 gene products. Transactivation occurred poorly or not at all when any one of these three transactivators was omitted. TRS1 is a member of the US22 family of proteins and is encoded by a region near the L-S junction of the viral genome within the c repeat and adjacent Us sequences. TRS1 is highly homologous to IRS1, which is encoded from the other copy of the c repeat, and plasmid constructs carrying the irs1 gene were also able to mediate transactivation of the ICP36 promoter. RNA blot analysis of steady-rate RNA throughout infection showed that the trs1 transcript was expressed with the kinetics of an alpha gene but its accumulation was delayed relative to that of ie1 and ie2 transcripts. On the basis of these experiments, TRS1 and IRS1 are proposed to be important intermediaries in the cascade of cytomegalovirus gene expression.