To investigate the expression of human immunodeficiency virus (HIV) genes in human monocytes, a DNA transfection system was developed and characterized using cultured primary monocytes. Monocytes that were cultured 6-7 days in an adherent monolayer were efficiently recovered and transfected by electroporation with an expression vector containing the Escherichia coli lacZ gene under control of the cytomegalovirus immediate-early promoter. Successful transfection was detected by expression of beta-galactosidase activity and by histochemical staining for beta-galactosidase in cells that were allowed to readhere to plastic following transfection. Over 30% of the surviving adherent monocytes expressed the transfected beta-galactosidase gene. In the same manner, monocytes were transfected with HIV provirus clones pIIIB and pIIB/PB. The provirus pIIIB/PB differs from pIIIB only in that it contains a small sequence from the env gene of a macrophage tropic HIV-1. Virus derived from pIIIB will not replicate in monocytes whereas virus derived from pIIIB/PB will. Monocytes transfected with either provirus DNA expressed high levels of p24 antigen within 1 day of transfection, and cell-free supernatants contained virus that was infectious for T cells. In contrast, only supernatants from pIIIB/PB transfections contained virus capable of infecting monocytes. Thus, proviral DNA of T cell tropic HIV efficiently completes the retroviral life cycle in monocytes in a manner indistinguishable from that of macrophage tropic HIV, and progeny virus retain their T cell tropism.