The metabolism of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) was studied using cultured fibroblasts deficient in acid β-glucosidase activity. In fibroblasts from patients with Gaucher's disease, in vitro β-glucosidase activities were 2.7-11.7% and 4.8-13.6% of control values when 4-methylumbelliferyl β-D-glucoside and GlcSph were used as substrates, respectively. In spite of the enzyme deficiency, GlcCer and GlcSph, the natural substrates of the deficient enzyme, did not accumulate in the cells. When normal fibroblasts were incubated with conduritol B epoxide (CBE), a specific inhibitor of acid β-glucosidase, the in vitro enzyme activities decreased dose-dependently (2.2-2.4% of control values at 50 μM CBE), and GlcCer and GlcSph accumulated in the cells at concentra-tions of CBE higher than 50 μM. To investigate the intracellular metabolism of GlcCer and GlcSph, either radioactive GlcCer or GlcSph was loaded onto cultured fibroblasts. In fibroblasts treated with a high dose of CBE (1mM), the degradation of GlcCer and GlcSph was retarded (5-21% on day 7; normal range, 81-99%), while in fibroblasts from patients with Gaucher's disease, both the pattern and rate of the degradation of the lipids (83-97% on day 7) were almost the same as those seen in the control cells. These results indicate that in Gaucher's disease fibroblasts the intracellular metabolism of GlcCer and GlcSph is normal in spite of the deficiency in β-glucosidase activity. At present it is not known why there is a discrepancy between the in vitro enzyme activity and the intracellular metabo-lism of the substrates, but the residual activity in the fibroblasts may well play a critical role in the in vivo degradation of the lipids in the fibroblasts, and these data may therefore explain the fact that the accumulation of GlcCer and GlcSph is confined to certain tissues in patients with Gaucher's disease.