We have characterized the effect of differentiation on the resistance of the mononuclear phagocyte to herpes simplex virus type 1 (HSV-1) by using the human mononuclear phagocyte cell line, U937. The replication of HSV-1 was compared in undifferentiated U937 cells and U937 cells induced to undergo differentiation. Undifferentiated U937 cells were highly resistant to HSV-1 infection. Infectious virus levels declined rapidly to less than 0.1 plaque-forming units (PFU) per cell by 24 hr postinfection and the cells were completely resistant to HSV-1-induced cytopathic effect. Differentiation of U937 cells by treatment with phorbol 12-myristate 13-acetate (PMA) was accompanied by a decrease in the resistance to HSV-1 infection. Infectious virus yields were increased greater than 75-fold at 24 hr postinfection, as compared with the undifferentiated U937 cells. PMA-differentiated U937 cells also acquired full susceptibility to HSV-1-induced cytopathic effect. Infectious center assay revealed that the percent of productively infected cells increased from approximately 3% in undifferentiated U937 to greater than 50% in PMA-differentiated cells. U937 cells were also induced to differentiate by treatment with all-trans-retinoic acid, dimethyl sulfoxide, and lymphokine as shown by differentiation-associated changes in morphology and cytochemical enzymes. These cells, however, failed to display increased permissiveness for HSV-1, indicating that the change in permissiveness was uniquely associated with PMA treatment. Undifferentiated U937 cells adsorbed as much virus as PMA-differentiated cells, but immunofluorescence assays, as well as DNA hybridization analysis demonstrated that an early block in HSV-1 replication occurred in undifferentiated U937 cells, before synthesis of viral protein and DNA. PMA-induced differentiation of U937 cells appears to release an early block in HSV-1 replication that is present in undifferentiated U937 cells.