Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the specific sulfonation of estrogens at the 3-hydroxyl position using 3'-phosphoadenosine-5'-phosphosulfate as an activated sulfate donor. Sulfated estrogens no longer bind to the estrogen receptor and are, therefore, hormonally inactive. Although liver has been considered a primary site for steroid sulfotransferase activities, we previously have cloned the mouse EST complementary DNA and found the enzyme to be expressed abundantly in the testis of normal mice. In this study we show by reverse transcription-PCR that EST is also expressed in the testes of rat and man, suggesting that testicular expression of EST may be a common phenomenon among different species. Using a purified polyclonal antibody raised against the bacterially expressed mouse EST protein, we demonstrate by immunohistochemistry that EST is localized selectively to the androgen-producing Leydig cells within the mouse testis. Additionally, we show that Leydig cell expression of EST is under the control of the pituitary hormone LH and is regulated differentially during development. In contrast to the high level of expression in mature intact animals, EST is not present in Leydig cells of hypophysectomized mice or in Leydig cells of fetal and prepubertal (day 5 or 17) mouse testes. Administration of hCG to hypophysectomized mice restored the testicular expression of EST. Together, these results suggest that testicular expression of EST may play an important role in male reproduction, conceivably by modulating the activity of locally synthesized estrogen in the testis of a sexually mature animal.