Human immunodeficiency virus (HIV-1) is the primary retroviral agent responsible for AIDS and related disorders worldwide. One of its identified gene products, tat protein, stimulates in trans the expression of all HIV-1 genes by several orders of magnitude. Cells infected with HIV-1 require tat protein to produce virus, suggesting that trans-activation is crucial for viral replication. The essential cis-acting site for trans-activation, termed tar, resides within the R region of the HIV-1 long terminal repeat (LTR), between -17 and +54 with respect to the initiation site of viral transcription. It is striking that the RNA encoded between +1 and +59 has the potential to form an extensive stem-loop secondary structure which, as a portion of the untranslated leader RNA, would be common to all HIV-1 mRNAs. We now present the results of nucleotide substitution experiments which suggest that tat trans-activation requires presentation of the sequence +30CUGGG+34 in tar within the loop of a RNA hairpin structure.