Prenatal development of the epididymis was studied in bovine fetuses ranging from 10 to 90 cm crown-rump length (CRL) (75-285 pcd). The studies aimed to apply both glycohistochemistry and immunohistochemistry for the detection of the differentiation of the developing prenatal epididymis. Both conventional histological and histochemical techniques were applied on paraffin sections of the epididymis from different fetal stages. Establishment of the urogenital junction between the extra-testicular rete testis and the mesonephric duct, via the growing efferent ductules (ductuli efferentes) was first evident in fetuses with 10 cm CRL. At the fetal age of 110 pcd (24 cm CRL), the mesonephric duct began to lengthen and coil forming three distinct regions (caput, corpus and cauda). In addition to the macroscopical modifications in the extra-testicular excurrent duct system, histological differentiation involved both the tubular epithelial and the peritubular mesenchymal cells. The epithelium lining the efferent ductules was differentiated into ciliated and non-ciliated columnar cells. The simple epithelium of the epididymal duct increased in height and developed stereocilia on the apical surface. Additionally, some basal cells first appeared at 185 pcd (56 cm CRL), within the epithelium lining the cauda only. Lectin histochemistry (WGA, PNA, GSA-I) showed early immunostaining in epithelium of the efferent ductules and in peritubular mesenchymal structures. Immunoreactivity for different proteins (S-100, fibroblast growth factor-1 and factor-2, angiotensin converting enzyme, laminin, alpha-smooth muscle actin) was evident, both in the epithelial and in the peritubular mesenchymal cells as early as at 75 pcd. On the basis of our histochemical observations, we conclude that both glycohistochemistry and immunohistochemistry are useful tools to demonstrate that the differentiation in the peritubular structures and efferent ductular epithelium begins earlier than other components. (c) 2011 Elsevier GmbH. All rights reserved.