The acidic phospholipid requirement of the predominant particulate β-glucosidase of mammalian spleen and liver was investigated using a series of N-acyl derivatives of dioleoyl phosphatidylethanolamine (PE). The PE, a neutral phospholipid, was converted to an acidic lipid, (N-acyl)-phosphatidylethanolamine (NAPE) by acylation of the amino group with different fatty acyl chains. Lysosomal β-glucosidases from rat liver and spleens of controls and patients with various types of Gaucher's disease were solubilized and delipidated by extraction with sodium cholate and 1-butanol. All members of the NAPE series tested were effective activators of the delipidated rat liver β-glucosidase, and the stimulatory power of the NAPE family increased with increasing chain length of the fatty acid substitution. In contrast, dioleoyl-PE had no effect on β-glucosidase activity. A heat-stable factor (HSF) purified from the spleen of a patient with Gaucher's disease significantly increased the sensitivity of the rat liver β-glucosidase to all of the NAPE derivatives. The maximum stimulation achieved in the presence of HSF was independent of N-acyl chain length. Compared to the potent activator, phosphatidylserine (PS), (N-acetyl)-PE and (N-linoleoyl)-PE were half as effective as activators of β-glucosidase from control human spleen. PS stimulated the β-glucosidase of type 1 nonneurologic Gaucher's disease, but none of the NAPE compounds activated it. Neither PS nor any of the (N-acyl)-PE compounds could activate a delipidated preparation of β-glucosidase obtained from the spleen of a neurologic case. These results indicate that although the presence of a net negative charge on a phospholipid confers upon it an ability to reconstitute β-glucosidase activity to the normal, nonmutant enzyme, it is insufficient to permit differentiation of the various types of Gaucher's disease.