In the human macrophage-like cell line U937, which is resistant to infection with herpes simplex virus type 1 (HSV-1), it was previously shown that resistance can be overcome by inducing differentiation of the cells by treatment with phorbol 12-myristate 13-acetate (PMA). The present data show that differentiation, and not PMA treatment alone, enabled HSV-1 replication, because vitamin D3 and mezerein were also able to cause U937 cells to differentiate to a state permissive for HSV-1 infection. Additionally, a portion of the undifferentiated cells underwent a productive infection when treated with PMA 2 days after infection, suggesting persistence of HSV-1 in these cells. The nonpermissiveness of the undifferentiated cells was further defined. Resistance did not involve differences in virus uptake, because the amounts of viral DNA in the infected cells and nuclei of differentiated and undifferentiated U937 cells were not significantly different early after infection. However, only very low levels of RNA for HSV-1 immediate-early, early, and late genes could be detected in the undifferentiated U937 cells by Northern blot analysis compared with the differentiated U937 cells. These data suggest that the primary block in HSV-1 replication in undifferentiated U937 cells occurred after transport of the viral DNA to the cell nucleus but prior to steady-state accumulation of viral RNA for immediate-early genes.