Aromatase activity has been measured in Leydig cells and Sertoli cells from both immature and mature rats. Cytochrome P450 aromatase (P450_arom) has been immunolocalized in germ cells of the rodent, bear, and rooster. Our purpose was to investigate expression of and to immunolocalize P450_arom in adult rat testicular cells. After Western blotting with a specific anti-cytochrome P450_arom antibody, we demonstrated the presence of a 55-kDa protein in mature rat seminiferous tubules and crude germ cell preparations. Immunoreactive aromatase was detected both in cultured rat Leydig cells and in testis sections (interstitial tissue and elongated spermatids showed positive immunoreactivity for P450_arom). We next used reverse transcription-polymerase chain reaction to localize and quantify the P450_arom mRNA in the various testicular cells. In rat Leydig cells, the amount of P450_arom mRNA was 15 times higher than in Sertoli cells (34.1 ± 3.2 to 2.3 ± 0.2 × 10^-3 amol/10⁶ cells, respectively). In pachytene spermatocytes, round spermatids, and testicular spermatozoa the P450_arom mRNA levels were 38.7 ± 8.1, 20.4± 3.8, and < 1.3 × 10⁻³ fmol/10⁶ cells, respectively. The aromatase activity was 2.5–4 times higher in testicular spermatozoa (8.48 ± 1.98 fmol/10⁶ cells per hour) than in other germ cells. These results indicate that in mature rats, not only Leydig cells and Sertoli cells but also germ cells have the capacity to express functional P450_arom. According to the germ cell maturation state, there was an inverse relationship between P450_arom mRNA content and the biological activity of the protein. The expression of the functional P450_arom in mature rat germ cells confirms the existence of an additional source of estrogens within the genital tract of the male.