Human cytomegalovirus (CMV) alters the growth and expression of human immunodeficiency virus-1 (HIV-1) in cell culture and may accelerate the course of AIDS in HIV-1 infected patients. CMV infection or the expression of the CMV immediate early genes has been shown to activate gene expression directed by the HIV-1 LTR. However, the cis-acting elements within the HIV-1 LTR that confer responsiveness to CMV have not been clearly delineated. We report on investigations in human fibroblasts designed to precisely map this signal. Our studies demonstrate that more than one nonoverlapping region of the HIV-1 promoter is capable of responding to CMV. Sequences 3' from -19(relative to the start of transcription) are dispensable for CMV responsiveness. We also show that in addition to immediate early region 2, immediate early region 1 is able to activate HIV-1 LTR-directed gene expression.