The degradation of globotriaosylceramide (GbO-se3Cer) by insect-cell derived recombinant human alpha-galactosidase (EC 3.2.1.22) was carried out in a detergent-free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both alpha-galactosidase and sphingolipid activator protein B (SAP-B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both alpha-galactosidase (0.24-24 mU/50 microliters assay) and SAP-B (0-5 micrograms/50 microliters assay). The enzyme reaction was not affected by SAP-A, SAP-C, or SAP-D. Therefore, our results indicate that only SAP-B is essential for the degradation of GbOse3Cer by alpha-galactosidase.