The activity of N-acetylglucosamine-6-phosphate deacetylase, a key enzyme in the pathway of N-acetylglucosamine catabolism, was measured in hepatocytes, Kupffer cells and sinusoidal endothelial cells from rat liver and cultured human skin fibroblasts. Kupffer cells and endothelial cells had similar high levels of deacetylase activity that were more than twice the level observed in fibroblasts. In contrast, hepatocytes had extremely low activity (several hundredfold less than Kupffer cells and endothelial cells). A major implication of deacetylase deficiency in hepatocytes is that N-acetylglucosamine generated as a result of the catabolism of complex carbohydrates in these cells cannot enter glycolysis and must be largely reused for the synthesis of plasma glycoproteins and other N-acetylglucosamine-containing macromolecules.