Continuous replication of human immunodeficiency virus type 1 requires the expression of the regulatory protein Rev, which binds to the Rev response element (RRE) and up-regulates the cytoplasmic appearance of singly spliced and unspliced mRNA species. It has been demonstrated that the murine protein YL2 interacts with Rev in vivo and modulates the activity of Rev (Luo, Y., Yu, H., and Peterlin, B. M. (1994) J. Virol. 68, 3850-3856). Here we show that the YL2 human homologue, the p32 protein, which co-purifies with alternative splicing factor ASF/SF2, interacts directly with the basic domain of Rev in vitro and that the Rev-p32 complex is resistant to high concentrations of salt or nonionic detergent. Protein footprinting data suggest that Rev interacts specifically with amino acids within the 196-208 region of p32. An analysis of the ternary complex, formed among p32, Rev, and RRE RNA, shows that Rev can bridge the association of p32 and RRE. Furthermore, we demonstrate that exogenously added p32 specifically relieves the inhibition of splicing in vitro exerted by the basic domain of Rev. Our data are consistent with a model in which p32 functions as a link between Rev and the cellular splicing apparatus.