MHC class I genes are potently repressed by HIV Tat, which transactivates the HIV LTR. Tat represses class I transcription by binding to complexes associated with a novel promoter element, consisting of Sp1-like DNA binding sites. Transcription by other Sp1-dependent promoters, such as MDR1 and the minimal SV40 promoters, is also repressed by Tat, whereas the human beta-actin promoter is neither activated by Sp1 nor repressed by Tat. Tat repression can be overcome by a strong enhancer element. Thus, the SV40 72 bp enhancer element confers protection from Tat-mediated repression on both the minimal SV40 promoter and the class I promoter. Surprisingly, Tat can activate the class I promoter in the presence of both the HIV TAR element and a strong upstream enhancer. These data demonstrate that Tat differentially affects Sp1-responsive promoters, depending on promoter architecture.