The mammalian genome encodes a family of lactate dehydrogenase (LDH) isozymes. Two of these, ldha and ldhb, are expressed ubiquitously. The ldhc gene is active only in the germinal epithelium during spermatogenesis. In our analysis of ldhc gene regulation, we found that a 60-base pair promoter sequence was sufficient for testis-specific expression in an in vitro transcription assay. To confirm these findings, a genomic fragment containing 100 base pairs overlapping the transcription start site was isolated and linked to the Escherichia coli lacZ gene. We report that this genomic fragment drives testis-specific expression in transgenic mice. We conclude that transcription of the transgene and possibly of the endogenous ldhc gene is restricted to leptotene/pachytene primary spermatocytes.