The lipid requirement of membrane-bound rat liver β-glucosidase was investigated using 4-methylumbelliferyl-β-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated - phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 μg) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 μg of PtdSer markedly stimulated β-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 μg) or -PtdSer (5 μg) provided maximum protection of β-glucosidase against heat (60°C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (T(0.5), 12 ± 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 μg) and HSF (t(0.5), 2.8 min). The combination of PtdSer (10 μg) and HSF (1.3 μg) lowered the K(m) for 4-methylumbelliferyl-β-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activators substances. The inclusion of PtdSer and HSF in the β-glucosidase assay medium lowered the K(i) of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I(0.5) of the enzyme for glucosylsphingosine from 89.4 to 7.6 μM. A study of log (V(max)/K(m)) versus pH indicated that the PtdSer-HSF pair creates the active site of β-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.