Transcriptional regulation in human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. We have used a site-specific cross-linking method based on 4-thiouracil (4-thioU) photochemistry to determine the conformation of TAR RNA and its interaction with Tat protein under physiological conditions. Three different TAR RNA constructs with a single 4-thioU residue at position 23, 38, or 40 were synthesized. Upon UV irradiation, 4-thioU at all three positions formed interstrand covalent cross-links in TAR RNA. Determination of cross-link sites by RNA sequencing revealed that 4-thioU at position 23 makes a direct contact with U40, while a 4-thoU at position 40 cross-links to C24 and C25, and at position 38, 4-thioU contacts G26 in TAR RNA. The addition of arginine did not alter the yield or the site of RNA-RNA cross-link. However, in the presence of Tat(38-72), UV irradiation of RNA modified with 4-thioU at position 23 or 38 resulted in RNA- protein cross-links, but no RNA-RNA cross-links were observed. 4-thioU at position 40 formed both RNA-RNA and RNA-protein cross-links in the presence of Tat(38-72). An intriguing finding of our studies was that a cross-linked TAR RNA with 4-thioU at position 40 retained specific Tat-binding activity. Our results establish four important conclusions about Tat-TAR structure. (1) U23 of free TAR RNA is in close contact with U40. (2) U40 is in close proximity to C24 and C25 both in free TAR RNA and in a complex with Tat. (3) Tat protein directly contacts U23, U38, and U40 in the major groove of TAR RNA. (4) Tat protein can recognize a TAR RNA structure containing an interrupted bulge which is formed by a covalent link between U40 and two bulge residues, C24 and C25. These structural studies provide new insights into tertiary folding of TAR RNA and its interaction with Tat protein.