Forced overexpression of TEAD1 in human uterine fibroblast (HUF) and human endometrial stromal cells markedly inhibited prolactin promoter activity in both cell types in a dose-dependent manner, with maximal inhibition of greater than 90%. Conversely, the knockdown of TEAD1 expression in HUF cells with a TEAD1 siRNA resulted in a 75-80% increase in prolactin mRNA levels (p<0.01) compared to control cells exposed to a scrambled nonsense RNA. Mutagenesis of the putative TEAD site inhibited basal promoter activity by about 80%. However, mutagenesis of the TEAD site did not prevent TEAD1-induced inhibition of promoter activity; and the transcription activity of a minimal promoter fragment lacking a putative TEAD binding site was repressed by overexpression of TEAD1. Taken together, these findings suggest that the TEAD binding site on the prolactin promoter is important for the maintenance of basal prolactin promoter activity and that overexpression of TEAD1 has a dominant-negative effect on prolactin promoter activity, probably by interacting directly with other transcription factors.