Large amounts of cathepsin L (CTSL), a cysteine protease required for quantitatively normal spermatogenesis, are synthesized by mouse and rat Sertoli cells during stages VI to VII of the cycle of the seminiferous epithelium. We previously demonstrated that all of the regulatory elements required in vivo for both Sertoli cell- and stage-specific expression of the Ctsl gene are present within a ~3-kb genomic fragment that contains 2065 nucleotides upstream of the transcription start site and 977 nucleotides of downstream sequence. Most of the downstream region encodes the first intron. In this study, transient transfection assays using primary Sertoli cell cultures and the TM4 Sertoli cell line established that the Ctsl first intron increased reporter gene activity by ~5-fold. While the intron-mediated enhancement in reporter gene activity was not restricted to the Ctsl promoter, positioning the first intron upstream of the Ctsl promoter in either orientation abolished its stimulatory activity, suggesting that it does not contain a typical enhancer. Mutating the 5′-splice site of the Ctsl first intron or replacing the first intron by the Ctsl fourth intron abolished the stimulatory effect. Finally, the intron-dependent increase in reporter gene activity could be explained in part by an increase in the amounts of total RNA and transcript polyadenylation. Results from this study suggest that the stimulatory effect mediated by the Ctsl first intron may explain in part why Sertoli cells in seminiferous tubules at stages VI to VII produce high levels of CTSL.