Previous experiments revealed that male transgenic mice bearing a cosmid that included the Class II E_α gene, about 35 kb of 5′ flanking DNA, and the cosmid vector sequences were sterile. To ascertain the cause of the sterility, various subfragments of the cosmid were tested in transgenic mice. Only those pieces of DNA that included some of the E_α flanking chromosomal DNA and the herpes simplex virus (HSV)-thymidine kinase (tk) gene that was in the vector resulted in male sterility. Histological analysis revealed abnormalities in nuclear morphology of elongating spermatids and retention of mature spermatids within the seminiferous epithelium. Immunocytochemical studies showed that the HSV-tk gene was expressed at low levels in postmeiotic round spermatids and at higher levels in more mature elongating spermatids. To determine whether expression of HSV-tk in spermatids might be responsible for the sterility, the protamine gene promoter was used to direct the expression of HSV-tk to postmeiotic germ cells. Since the mice so treated were also sterile, the data suggest that expression of this enzyme in spermatids is responsible for the sterility phenotype.