We have purified a 52 kd protein, AP-2, that binds to enhancer regions of SV40 and human metallothionein IIA (hMT IIA) and stimulates RNA synthesis from these promoters in vitro. Surprisingly, AP-2 also binds to two SV40 early promoter regions recognized by Spl and T antigen. Juxtaposed binding sites for AP2 and Spl in the 21 bp repeats may facilitate productive interactions between the two factors. In contrast, sequencespecific binding of AP-2 to SV40 and hMT IIA DNA is inhibited by the viral repressor protein T antigen. Furthermore, T antigen inhibits AP-a-dependent transcriptional activation of the hMT IIA promoter in vitro. The inhibition is neither a direct nor an indirect result of T antigen binding to DNA, because the hMT IIA promoter lacks T antigen binding sites. Instead, sedimentation studies suggest that protein-protein interactions between AP2 and T antigen block AP-2 binding to DNA. These findings suggest novel mechanisms for mediating positive and negative regulation of transcription.