Estrogen is found in high concentrations in the excurrent duct were it regulates the expression of genes involved in water reabsorption. Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes specific sulfonation with a high affinity for estrogens. Since sulphated estrogens do not bind to estrogen receptors, they are considered to be hormonally inactive. EST may thus determine where along the male tract estrogenic environment predominates. Sulfotransferase activity increases along the epididymis and may also play a role in sperm physiology during the epididymal transit. Using the bovine as a model, we investigated the distribution of EST along the excurrent duct and the possibility that sterols associated to spermatozoa can be substrates of this enzyme. RT-PCRs showed that mRNA encoding EST was expressed in the testis and all along the epididymis. A highly specific antiserum was raised against the bovine recombinant EST and used in western blots and immunohistological studies. Western blots of tissue homogenates showed that EST was localized all along the excurrent duct with a higher signal in the caput and corpus epididymidis. EST was detectable in the intraluminal compartment only in the caput epididymidis where it was associated to epididymosomes and spermatozoa. EST was undetectable in different fraction of fluids collected in the cauda segment. In immunohistological studies, EST was restricted to the acrosomal region of the caput but not the cauda epididymal spermatozoa and detectable in the cytoplasm of the epithelium bordering the lumen all along the epididymis as well as in the rete testis and vas efferens. This enzyme was also associated to the nucleus in the caput and corpus as well as with the apical membrane of the corpus epididymal epithelium. When recombinant EST was incubated in vitro in presence of caput and cauda spermatozoa, this enzyme was able to sulphate sperm membrane cholesterol. Our study shows that EST is present in both the intracellular and intraluminal compartments of the epididymis, suggesting that this enzyme plays different roles along the excurrent duct.