The visna virus Tat protein is a strong transcriptional activator and is necessary for efficient viral replication. The Tat protein regulates transcription through an AP-1 site proximal to the TATA box within the viral long terminal repeat (LTR). Previous studies from our laboratory using Tat-Gal4 chimeric proteins showed that Tat has a potent acidic activation domain. Furthermore, a region adjacent to the Tat activation domain contains a highly conserved leucine-rich domain which, in the context of the full-length protein, suppressed the activity of the activation domain. To further elucidate the role of this region, four leucine residues within this region of Tat were mutated. In transient-transfection assays using visna virus LTR-CAT as a reporter construct, the activity of this leucine mutant was dramatically reduced. Additionally, domain-swapping experiments using the N-terminal activation domain of VP16 showed that the leucine-rich domain of Tat confers AP-1 responsiveness to the chimeric VP16-Tat protein. A chimeric VP16-Tat construct containing the leucine mutations showed no increased AP-1 responsiveness in comparison with that of the VP16 activation domain alone. Furthermore, in competition experiments, a Gal4-Tat protein containing only the leucine region of Tat (amino acids 34 to 62) was able to inhibit by competition the activity of full-length Tat. These studies strongly suggest that this leucine-rich domain is responsible for targeting the Tat protein to AP-1 sites in the viral LTR. In addition, examination of the amino acid sequence of this region of Tat revealed a highly helical secondary structure and a pattern of residues similar to that in the leucine zippers in the bZIP family of DNA-binding proteins. This has important implications for the interaction of Tat with cellular proteins, specifically Fos and Jun, that contain bZIP domains.