The dhod gene encodes dihydroorotate dehydrogenase (DHOdehase), which catalyzes the fourth step of de novo pyrimidine biosynthesis. In addition to the common 1.5 kb dhod RNA expressed by embryos and females, adult males produce a group of slightly longer RNAs. Evidence is presented that the latter RNAs arise through transcription initiation at sites upstream from that of the common RNA and expression of these male-specific RNAs is limited to spermatogenesis. In situ hybridization analysis shows that these RNAs accumulate during spermatocyte growth and persist through meiosis and early spermatid differentiation. In contrast, DHOdehase activity is virtually absent in spermatocytes, meiotic cells, and in early spermatid cysts, then it becomes highly abundant in elongated spermatid cysts and disappears in late spermatogenesis. Thus, testis-limited expression of dhod conforms to a model proposed for other genes that function during spermiogenesis: transcription in spermatocytes, storage of translationally inactive RNA through meiosis, translation of the RNA during spermiogenesis. Very similar expression of a testis promoter-lacZ fusion transgene indicates that sequences required for the spermatogenesis transcription and translation patterns are confined to the 5' end of the dhod gene. Deletion analysis of that 5' region delimits all sequences necessary for spermatid expression of the transgene to a 89 bp fragment. These results are discussed in the contexts of known mechanisms of gene regulation during spermatogenesis and potential roles of DHOdehase during spermiogenesis.