The differentiation of U937 monoblastoid cells after human immunodeficiency virus type 1 (HIV-1) infection was studied using the following approaches: reverse transcriptase activity measurement, immunofluorescence labeling, and electron microscopy. For comparison, uninfected U937 cells were induced to differentiate from monocyte to macrophage by phorbol 12-myristate 13-acetate (PMA) or retinoic acid (RA) treatment. Both infected and drug-treated cells showed important and similar ultrastructural cell modifications, with a phenotype that decreased in monocyte specificity and increased in that of macrophages. When U937 cells were induced to differentiate upon HIV-1 infection, a very different pathway of viral production was observed. Production and accumulation of the virus in a vacuolar compartment of intracytoplasmic origin and escape to the antiviral lysosomal activity could explain virus persistence. This makes the cell system a good model with which to study the relationship between HIV-1 production and cell differentiation.