We have cloned a murine transforming growth factor (TGF) beta-3 complementary (cDNA) from normal tissue by low stringency screening of a testicular cDNA library with a TGF-beta-1 probe. The coding domain of this TGF-beta-3 cDNA agrees completely with the sequence reported for the TGF-beta-3 cDNA isolated from the AKR-2B cell line, but the testicular clone uses a distinct and unusual polyadenylation signal resulting in an altered 3' untranslated domain. Northern blot hybridization analysis of gonadal tissues showed that both TGF-beta-3 and TGF-beta-1 mRNAs are detectable in the mouse testis and ovary. A detailed analysis of TGF-beta-3 and TGF-beta-1 gene expression in normal and germ cell-deficient male mice showed that the somatic cell compartment of the mouse testis expresses the usual-sized transcripts for both genes. However, a smaller (1.8-kilobase) TGF-beta-1 mRNA is expressed selectively in male germ cells, and expression of this transcript was constitutive throughout the spermatogenic stages examined. This result demonstrates a new pattern of TGF-beta-1 gene expression, consistent with cell lineage-specific transcriptional regulation during spermatogenesis.