Properties of glucosylsphingosine (gluco-psychosine) glucosyl hydrolase were studied in detail in cultured human fibroblasts and placenta and were compared with those of glucosylceramidase. The two activities, that are deficient in tissues of Gaucher patients, showed minor but consistent differences. The pH optima were 4.8 for psychosine hydrolysis and 5.3 for glucosylceramide hydrolysis. In the presence of oleic acid, taurocholate activated glucosylceramidase more than 10-fold, while it activated psychosine hydrolysis only by about 30%. Triton X-100 was stimulatory for glucosylceramidase but was strongly inhibitory for psychosine hydrolysis. Phospholipids, that increase many times glucosylceramidase activity, were moderately inhibitory to enzymatic hydrolysis of psychosine. The psychosine hydrolase activity was slightly more heat-stable than the glucosylceramidase activity. The Km values for the two substrates were similar; 1.7 X 10(-5) M for psychosine and 2.7 X 10(-5) M for glucosylceramide. The V for glucosylceramide was, however, 100-times that for psychosine hydrolysis. Psychosine acted as a potent non-competitive inhibitor (Ki = 1.8 X 10(-5) M), while glucosylceramide was a weak inhibitor against psychosine hydrolysis. Within the limit of glucosylceramide solubility, psychosine hydrolysis could not be inhibited by more than 50%. Furthermore, the Dixon plot of glucosylceramide inhibition showed an anomalous slope. The ratio of the two activities was similar in fibroblasts, in the placenta mitochondria-lysosomal fraction and in a partially purified placental preparation. These findings are best explained by the hypothesis that, although the two substrates are hydrolyzed by a single enzyme, they share an overlapping but not identical catalytic site while binding to hydrophobic sites unique for the respective substrates.