Aromatase activity has been measured in Leydig cells and Sertoli cells from both immature and mature rats. Cytochrome P450 aromatase (P450arom) has been immunolocalized in germ cells of the rodent, bear, and rooster. Our purpose was to investigate expression of and to immunolocalize P450arom in adult rat testicular cells. After Western blotting with a specific anti-cytochrome P450arom 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 P450arom). We next used reverse transcription-polymerase chain reaction to localize and quantify the P450arom mRNA in the various testicular cells. In rat Leydig cells, the amount of P450arom 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 P450arom 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 P450arom. According to the germ cell maturation state, there was an inverse relationship between P450arom mRNA content and the biological activity of the protein. The expression of the functional P450arom in mature rat germ cells confirms the existence of an additional source of estrogens within the genital tract of the male.