Abstract Previous studies have demonstrated that the induction of an estrogen-regulated protein precedes the desensitization of microsomal enzymes after in vitro treatment of Leydig cells with hCG or estradiol (E 2). This protein is recognized by a monoclonal antibody against an estrogen-dependent protein of mol. wt 28,000 from MCF-7 cells [ Science 226 (1984) 445]. In the present study, this antibody was used to investigate the ontogenesis of this protein, to analyze its regulation after hormonal treatments under in vitro conditions and to determine its subcelluar localization. The study was performed at light and EM levels using immunocytochemical techniques. The estrogen-regulated protein was detectable with low reactivity in the fetal testis and was undetectable from birth to 20 days. During the latter period, Leydig cells are in a quiescent phase of their maturation, with low aromatase activity and low estrogen and testosterone production. Thereafter, increasing immunostaining from 20 to 60 days was observed concurrent with Leydig cell maturation. Reactivity was constant throughout adulthood. The in vitro studies demonstrated that the synthesis of the protein in culture of adult Leydig cells is stimulated by hCG via the action of endogenous estrogen and is prevented by pretreatment with tamoxifen. The presence of the specific immunoreactive protein in E 2-treated Leydig cells and uterine cells from E 2-treated animals was also demonstrated by Western blot analysis. This is a long-lived protein, and 3 h of E 2 stimulation are required to produce an immunocytochemical change. Pretreatment with cycloheximide prevented the increase in synthesis induced by E 2. At the EM level (specific antibody/protein A-gold), the protein was detected in moderate amount mainly in the cycloplasmic matrix and close to the cisternae of the rough and smooth endoplasmic reticulum. Unlike the MCF-7 cell protein, it is not associated with secretion granules. The low reactivity observed in fetal cells is attributable to maternal estrogen affecting a small pool of estrogen receptors that is not sufficient to mediate the regulation observed in adult rats. Assessment of the estrogen-regulated protein by immunocellular techniques provides a sensitive index of estrogen receptor mediated action. This protein could be involved in intracellular modulatory function(s) in the Leydig cell.