Two novel fluorescent glycolipids, LRO-glucosylceramide (LRO-GC) and LRO-trihexosylceramide (LRO-THC) were synthesized and utilized for estimating activities of the lysosomal, acid beta-glucosidase in cell extracts and intact skin fibroblasts, derived from normal individuals and patients with Gaucher disease subtypes. The uniqueness of the glycolipids is the fact that a fluorescent probe (lissamine rhodamine) is linked in a sulfonylamide linkage to the sphingosyl residue of the sphingolipid. Thus, the product of enzymatic hydrolysis, lissamine rhodamine sulfonylamido sphingosine (LRO-ceramide) cannot be further hydrolyzed and remains a metabolic end product. A unique property of LRO-GC as a substrate for the lysosomal, acid beta-glucosidase in vitro was the observation that enzymatic hydrolysis occurs in the absence of detergents and that hydrolytic rates are, in fact, reduced in the presence of Triton X-100 and/or sodium taurocholate. Also, both glycolipids penetrated the membrane of intact fibroblasts in the absence of serum and were hydrolyzed in lysosomes of the intact cells. The rates of intracellular hydrolysis decreased with the severity of the Gaucher disease subtypes. Using LRO-THC as substrate, the intracellular ratio of LRO-ceramide to LRO-glucosylceramide was an indicator for the specific GD-subtype.