Replication of human immunodeficiency virus type 1 requires expression of the viral trans activator Rev. Rev binds to a highly structured RNA, the Rev response element, which is present in singly spliced and unspliced genomic viral RNAs. Although Rev helps to transport these transcripts from the nucleus to the cytoplasm, the mechanism(s) involved is not fully understood. Using the yeast two-hybrid system, we isolated a murine protein (YL2) that interacts with the basic domain of Rev, which is essential for the function of Rev in vivo and for the inhibitory splicing activity of Rev in vitro. YL2 has 92% identity to a human 32-kDa protein (p32), which copurifies with alternative splicing factor SF2/ASF. Furthermore, we found that whereas expression of YL2 greatly potentiated the activity of Rev, antisense YL2 transcripts blocked the effects of Rev in mammalian cells. YL2 also increased the activities of Rex on the Rex response element and of hybrid Rev proteins fused to Tat and the coat protein of bacteriophage MS2 on their respective RNAs. Thus, YL2 or p32 is a cellular protein that modulates the function of human immunodeficiency virus type 1 Rev.