It has been proposed that stage-specific gene expression in Sertoli cells results from sequential activation and repression of transcription. However, the exact molecular mechanisms are unknown. As a first step in addressing this fundamental issue, we recently demonstrated that a 3-kilobase (kb) genomic fragment immediately upstream of the rat cathepsin L translation start site directed stage-specific expression of a reporter gene only in Sertoli cells of transgenic mice in a manner comparable to that of the endogenous gene (predominantly in stages VI–VIII tubules). Supporting the activation/repression model of regulation, an upstream domain that mediated an inhibitory effect by male germ cells was identified within this 3-kb promoter region. In the present study, we localized and characterized the regulatory elements that activate transcription. Analyses of a series of 5′ deletion constructs demonstrated that a 120-base pair (bp) region that spans the transcription start site of the rat cathepsin L gene was sufficient to activate transcription in Sertoli cells isolated from sexually mature rats. Within this region, electrophoretic mobility shift assays showed that one member of the Sp/XKLF family of factors, Sp3, specifically bound to a GC-box. Furthermore, Sp1-binding activity was not detected in nuclear extracts from Sertoli cells of sexually mature rats. Finally, the GC-box was shown to be essential for promoter activity since mutating this binding motif abolished promoter activity. Collectively, these results suggest that the GC-box is a critical regulatory element for the cathepsin L promoter in mature Sertoli cells.