Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of beta-galactosidase and beta-glucosidase. To determine the presence of beta-galactosidase and beta-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.