The present work describes a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Rev protein and the cellular lens epithelium-derived growth factor p75 (LEDGF/p75) protein in vitro and in virus-infected cells. Here we show, for the first time, that formation of an Rev-LEDGF/p75 complex is a crucial step in regulating viral cDNA integration. Coimmunoprecipitation experiments at various times after virus infection revealed that, first, an integrase enzyme (IN)-LEDGF/p75 complex is formed, which is then replaced by a Rev-LEDGF/p75 and Rev-IN complexes. This was supported by in vitro experiments showing that Rev promotes dissociation of the IN-LEDGF/p75 complex. Combination of the viral IN and the cellular LEDGF/p75 is required for proper integration of the viral cDNA into the host chromosomal DNA. Our findings demonstrate that integration of HIV-1 cDNA is regulated by an interplay between viral Rev and the host-cell LEDGF/p75 proteins.