Ceramide, an intracellular lipid mediator of tumor necrosis factor alpha (TNF-alpha) action, was studied for its effects on the expression of the proviral human immunodeficiency virus type 1 genome in latently infected myelomonocytic cell lines U-1IIIB and OM-10.1. Ceramide treatment resulted in a 20- to 100-fold enhancement of HIV production in these cells. Ceramide also enhanced the expression of the chloramphenicol acetyltransferase gene directed by a human immunodeficiency virus type 1 long terminal repeat in transfected U-937 cells, indicating that ceramide acts at the level of viral transcription. These observations suggest that the TNF-ceramide signaling system may be involved in the regulation of HIV expression in certain myeloid cell types.