Northern blotting, in situ hybridization, and oligodeoxyribonucleotide excess solution hybridization were used to quantitate metallothionein-I (MT-I) and MT-II mRNAs in mouse testes. Testes from sexually mature adults contained high levels of both MT mRNAs (approximately 10-fold higher than those in control adult liver). Testicular MT mRNA levels were age dependent, being low the first 2 weeks after birth and increasing slowly thereafter to maximal levels in the adult (by 9 weeks after birth). In the adult testis, in situ hybridization indicated that only cells within the adluminal compartment (germ cells) of the seminiferous tubules contain high levels of MT mRNA. The appearance of cells containing elevated levels of MT mRNA during development was delayed from the onset of spermatogenesis. In situ hybridization suggested that MT mRNA accumulates after the initial differentiation of primary spermatocytes and is maintained in spermatids. Pachytene spermatocytes (PSC) and round spermatids (RTD) isolated from adult testes contained both MT-I and MT-II mRNAs in levels equivalent to those found in zinc-treated hepatocytes, whereas very low levels of MT mRNA were detected in isolated Sertoli cells (ST). In situ hybridization suggested that MT mRNA was present at only basal levels in interstitial, spermatogonial, and mature sperm cells at all developmental stages examined. Northern blot and in situ hybridization to sulfated glycoprotein-2 (SGP-2) mRNA, a ST-specific transcript, showed that SGP-2 mRNA is high in the testis of 1-week-old mice and decreases gradually to a lower level in the adult. In situ detection of this mRNA was consistent with the location of ST in the testis. SGP-2 mRNA was abundant in ST and rare in PSC and RTD preparations. Analysis of pulse-labeled proteins from isolated PSC and RTD indicated that these cells actively synthesize MT-I and MT-II. The high levels of MT mRNA in adult testes were not increased substantially after systemic injection of cadmium, zinc, or bacterial lipopolysaccharide. In marked contrast, these treatments led to dramatically increased levels of hepatic and ovarian MT mRNA. This study establishes that the MT genes are actively expressed in a developmentally regulated fashion in the male germ cells of the mouse. This suggests a role for MT in the process of spermatogenesis.