Glycosidases in rat epididymal fluid are secreted under androgen stimulation and possess receptors on the sperm surface. One of these enzymes, beta-D-galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). beta-D-Galactosidase was specifically localized to a subpopulation of larger, electron-dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classical fluorometric assay (used in previous papers) and colloidal gold (20 nm) conjugated to gal as a marker in ultrastructural studies. beta-D-Galactosidase bound to mbv with high-affinity (Kd in a nanomolar range) are in a saturable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a sugar that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, electron-dense mbv but not on the small, electron-lucent mbv. Gold particles were also observed on the larger vesicles, but less frequently in the presence of f-1.6-dip. Larger mbv possesses high-affinity sites for gal on their membrane.