Testes-specific protease 50 (TSP50) has been identified as one of the testis-specific proteins that is expressed at high levels in approximately 92% of human breast cancer samples, making it an attractive molecular marker and a potential target for diagnosis and therapy. However, little is known about the transcriptional mechanisms controlling TSP50 gene expression. In the present study, we have characterized the 5' regulatory region of the TSP50 gene in order to understand the molecular mechanisms regulating its expression. Analysis with a series of deletions demonstrated that a 624-bp region was essential for the basal promoter activity of the TSP50 gene. Further analysis results indicated that the two fragment regions +231 to +251 and -22 to -8, especially the putative Sp1 binding site (+237 to +239) and the putative CCAAT/enhancer binding protein (C/EBP) binding site (-15 to -13), are more important for the basal transcription activity of the human TSP50 promoter. Overexpression of Sp1 and C/EBPbeta transcriptional factors upregulated the activities of the TSP50 promoter. Taken together, these results will help to better understand the role of the TSP50 gene in signal-dependent transcriptional regulation, and to develop new reagents for therapeutic downregulation of the TSP50 gene in human breast cancer.