KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.