We have investigated the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and several potent vitamin D3 analogs [1,25(OH)2-16-ene-23-yne-D3; 1,25(OH)2-16-ene-23-yne-26,27-F6-D3] on productive infection by human immunodeficiency virus (HIV) in human macrophages. Macrophages derived from the peripheral blood were either pretreated with the vitamin D3 analogs, washed, and exposed to HIV (pre-infection treatment) or were infected with HIV, washed, and cultured with the vitamin D3 compounds (post-infection treatment). After three days of HIV-infection, levels of p24 antigen were measured. Pretreatment of macrophages with either 1,25(OH)2D3 or 1,25(OH)2-16-ene-23-yne-26,27-F6-D3 (pre-infection treatment) increased productive HIV infection about 3.5-fold; 1,25(OH)2-16-ene-23-yne-D3 increased levels about 4.7-fold. In contrast, exposure of HIV infected macrophages to the vitamin D3 compounds (post-infection treatment) did not affect levels of HIV production compared to untreated controls. Soluble CD4 completely inhibited productive HIV infection of macrophages pretreated with vitamin D3 analogs. Also, the vitamin D3 compounds slightly decreased CD4 expression on macrophages. The mechanism of enhanced productive HIV infection by the vitamin D3 compounds is unclear, but can not be explained by either alteration of CD4 expression or entry into cells by a CD4-independent route. These studies may have implications for both the basic biology of HIV infectious production and possibly clinical treatment of AIDS patients.