Based upon a prior study which evaluated a series of nonnucleoside pyrrolo[2,3-d]pyrimidines as inhibitors of human cytomegalovirus (HCMV), we have selected three active analogs for detailed study. In an HCMV plaque-reduction assay, compounds 828, 951, and 1028 had 50% inhibitory concentrations (IC50s) of 0.4 to 1.0 μM. Similar results were obtained when 828 and 951 were examined by HCMV enzyme-linked immunosorbent assay (IC50s = 1.9 and 0.4 μM, respectively) and when 828 was tested in a viral DNA-DNA hybridization assay (IC50 = 1.3 μM). In yield-reduction assays with a low multiplicity of infection (MOI), all three compounds caused multiple log10 reductions in virus titer, and the activities of these compounds were comparable to the activity of ganciclovir (GCV; IC90 = 0.2 μM). In contrast to the reduction of viral titers by GCV, the reduction of viral titers by 828, 951, and 1028 decreased with increasing MOI. Cytotoxicity in human foreskin fibroblasts and KB cells ranged from 32 to >100 μM. In addition, 828 (the only compound tested) was less toxic against human bone marrow progenitor cells than GCV. Time-of-addition and time-of-removal studies established that the three pyrrolopyrimidines inhibited HCMV replication before GCV had an effect on viral DNA synthesis but after viral adsorption. Compound 828 was equally effective against GCV-sensitive and GCV-resistant HCMV clinical isolates. Combination studies with 828 and GCV showed that the effects of the two compounds on HCMV were additive but not synergistic. Taken together, the data indicate that these pyrrolopyrimidines target a viral protein that is required in an MOI-dependent manner and that is expressed early in the HCMV replication cycle.