Past research has implicated both the Leydig and Sertoli cells as sources of testicular estrogens. To study this issue further, we investigated the ability of Sertoli cells isolated from the testes of immature (10‐day‐old) rats to synthesize estrogens in response to FSH in vitro. The aromatase activity of Sertoli cells was examined by comparing simultaneously two different methods: 1. the conversion of 19‐(6,7‐3H)‐hydroxyandrostenedione to (3H)estrone and (3H)estradiol, measured by silica gel thin layer chromatography; and 2. the conversion of unlabeled 19‐hydroxyandrostenedione to total estrogen (El + E2), measured by RIA.Sertoli cells, cultured in the presence of substrate, exhibited aromatase activity in response to FSH. Values obtained for total estrogen synthesis, measured by direct tracer methodology, at FSH dosages of 0.5 μg/ml and 1.0 μg/ml, during a 24‐hour period of incubation, were 620 pg/mg and 580 pg/mg, respectively; and after a 48‐hour incubation, values obtained were 570 pg/ml and 560 pg/mg, respectively. When measured by RIA, similar results were obtained for estrogen synthesis at identical FSH dosages and times of incubation. A dose response of FSH‐stimulated aromatization was also obtained by RIA. Neither control nor LH‐treated cells, however, in the presence of substrate, produced detectable levels of labeled or immunoassayable estrogens.Aromatase activity was also studied in the presence of a cold estrogen trap and measured by direct tracer methodology. In this case, estrogen synthesis was more than doubled. These data may suggest the further metabolism of estrogen by Sertoli cells. Finally, our results are discussed with respect to the role of the immature Sertoli cell in the synthesis of testicular estrogens.