Pem is a member of the homeobox transcription factor family that is expressed in somatic cells in male and female reproductive tissues. In the murine testis, Pem is specifically expressed in Sertoli cells, where it is dramatically induced at the initiation of meiosis during the first wave of spermatogenesis and then later is restricted to stages IV-VIII of the seminiferous epithelial cycle. To study the function of Pem in Sertoli cells, we generated transgenic mice that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in a large increase (6-fold) in the number of preleptotene spermatocytes with double-strand DNA breaks and a modest increase (2.5-fold) in the number of elongating spermatids (steps 9 and 10) with single-strand DNA breaks, based on terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick end labeling and comet analysis. The average number of DNA strand breaks in these germ cell populations also increased. Despite the transient increase in DNA strand breaks, Pem transgenic mice exhibited no significant anomalies in spermatogenesis, fertility, or fecundity. Our results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells.