Cultured endometrial stromal cells were susceptible to productive human cytomegalovirus (HCMV) infection. Infection of endometrial stromal cells resulted in pronounced cytopathic effects including cell rounding and aggregation, fusions, and some lysis, although not in the synchronous fashion observed in infected fibroblasts. The aggregation events were reminiscent of normal endometrial stromal cell responses to cyclical estrogen/progesterone levels. Immunofluorescence analysis demonstrated expression of viral gene products suggesting a productive virus infection. One-step growth analysis showed that infectious virus was produced but the titers were two logs lower than those obtained in fibroblasts even though HCMV DNA accumulated to similar levels in both cell types. In contrast, viral DNA replication was greatly reduced in endometrial stromal cells immortalized with a temperature-sensitive SV40 large T gene at both permissive and nonpermissive temperatures. A more detailed analysis of viral gene expression by Northern blotting revealed earlier appearances and greater initial levels of viral transcripts in endometrial stromal cells. No HCMV gene expression was observed at 120 hpi in these cells even though half of the cells were still intact and cellular gene expression was functional. Since this was the time of peak virus production, it seems plausible that reduced viral gene expression at late times p.i. was a major contributor to the reduced titers observed in endometrial stromal cells. These in vitro results coupled with in vivo observations by others of endometritis associated with HCMV suggest that further investigation into the effects of HCMV on the endometrium is warranted.