It has been shown that lysosomes are involved in B cell apoptosis but lysosomal glycohydrolases have never been investigated during this event. In this study we determined the enzymatic activities of some lysosomal glycohydrolases in human tonsil B lymphocytes (TBL) undergoing in vitro spontaneous apoptosis. Fluorimetric methods were used to evaluate the activities of beta-hexosaminidases, alpha-mannosidase, beta-mannosidase, alpha-galactosidase, beta-glucuronidase and alpha-fucosidase. Results show that in TBL during spontaneous apoptosis, there is a significant increase in the activity of beta-hexosaminidases, alpha-mannosidase, beta-mannosidase and beta-galactosidase. Also beta-glucuronidase and alpha-fucosidase activities increase but not in a significant manner. Further studies on beta-hexosaminidases revealed that also mRNA expression of the alpha- and beta-subunits, which constitute these enzymes, increases during spontaneous TBL apoptosis. When TBL are protected from apoptosis by the thiol molecule N-acetyl-L-cysteine (NAC), there is no longer any increase in glycohydrolase activities and mRNA expression of beta-hexosaminidase alpha- and beta-subunits. This study demonstrates for the first time that the activities and expression of some lysosomal glycohydrolases are enhanced in TBL during spontaneous apoptosis and that these increases are prevented when TBL apoptosis is inhibited.