Monocytotropic human immunodeficiency virus type 1 (HIV-1) isolates from patients with acquired immunodeficiency syndrome (AIDS) infect mononuclear phagocytes as well as activated T cells, but do not usually infect immature human myeloid cell lines in vitro. The HL-60 promyelocytic/myeloblastic cell line and the promonocytic line, U937, were susceptible to productive infection by monocytotropic HIV-1 isolates (HIV-1JR-FL and HTLV-IIIBa-L) after treatment with retinoic acid, dimethyl sulfoxide, dibutyryl cAMP, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Virus production was only detected when these compounds were added before virus infection. Virus replication did not correlate with CD4 receptor expression because undifferentiated HL-60 cells express CD4 and the level of CD4 expression did not increase after differentiation in the presence of retinoic acid, 1,25(OH)2D3, or TPA. A mature monocytic cell line (THP-1) was capable of infection without pretreatment, and treatment with differentiating agents enhanced virus production. A chronically infected cell line (J-HL-60) was isolated after HIV-1JR-FL infection of HL-60 cells treated with retinoic acid. Virus production in this cell line was enhanced more than 10-fold after differentiation in the presence of 1,25(OH)2D3 or TPA. The majority of virus production by 1,25(OH)2D3-treated J-HL-60 cells was associated with the mature, adherent population. Molecular analysis of a cloned line of J-HL-60 showed integration of a single DNA provirus. These results suggest that cellular factors associated with precursor cell differentiation along the myelomonocytic pathway are required for optimal replication of monocytotropic HIV-1 strains in vitro.