Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a stem-loop structure containing two helical stem regions separated by a trinucleotide bulge. The Tat protein contains a basic RNA-binding region (amino acids 49-57) located in the carboxyl-terminal half of the protein, and peptides containing this basic domain of Tat protein can bind TAR RNA with high affinities. We synthesized a 31-amino acid Tat fragment (amino acids 42-72) containing the basic region and part of flanking regulatory core domain that formed a specific complex with TAR RNA. Upon UV irradiation (254 nm), this Tat fragment cross-linked covalently with TAR RNA. Sites of cross-links were determined on both the TAR RNA and Tat protein fragment by RNA and protein sequencing, respectively. These results revealed that guanosine 26 of TAR RNA was cross-linked with tyrosine 47 of the Tat peptide. Our results provide the first physical evidence for a direct amino acid-base contact in Tat-TAR complex. Recently, orientation of the Tat-(42-72) was determined in our laboratory by psoralen.Tat-(42-72) conjugate (Wang, Z., and Rana, T. M. (1995) J. Am. Chem. Soc. 117, 5438-5444). On the basis of our findings, we suggest a model in which Tat binds to TAR RNA by inserting the basic recognition sequence into the major groove with an orientation where lysine 41 in the core domain of Tat contacts the lower stem and Tyr47 is close to G26 of TAR RNA. The knowledge of the orientation of Tat and details of other interactions with TAR RNA in Tat-TAR complex has significant implications for understanding gene regulation in HIV-1.