The activity levels of beta-N-acetylglucosaminidase, beta-N-acetylgalactosaminidase, beta-galactosidase and beta-glucosidase were fluorometrically assessed in spermatozoa, principal cells, basal cells and fibroblasts isolated from the rat epididymis by centrifugal elutriation. Among the various cell types, corpus principal cells had the highest activities for beta-N-acetylglucosaminidase, beta-N-acetylgalactosaminidase and beta-galactosidase. These enzymes characteristically react with membrane structural carbohydrates. Corpus/caput principal cell activity ratios of these glycosidases remained constant when determinations were done at an alternate pH and substrate concentration, suggesting that similar enzyme forms were present in both regions. Based on cell number and cell volume, sperm glycosidase activities generally increased from the caput to the corpus region of the epididymis, while decreasing from corpus to cauda. However, when data were expressed on the basis of cell protein, sperm glycosidase activities increased from caput to cauda. Since the total protein of sperm decreases dramatically from caput to cauda, the increase in glycosidase activity based on total protein suggests that relative to other sperm proteins, glycosidases may be selectively retained or taken up during epididymal transit. High levels of glycosidase activity associated with the corpus epididymidis may contribute to modification of sperm glycoproteins and observed increases in fertility of sperm as they emerge from this region.