Saposin B is a lysosomal sphingolipid-activator-protein which activates GM1-ganglioside hydrolysis by lysosomal beta-galactosidase. To identify the structural elements of saposin B implicated in sphingolipid binding, we studied a synthetic peptide corresponding to a predicted alpha-helix, sapB-18, spanning residues 52 to 69 of saposin B. The circular dichroism spectrum of sapB-18 at pH 4.4 was consistent with a 44% alpha-helix content. As shown by intrinsic Tyr fluorescence studies of sapB-18, this peptide binds the GM1-ganglioside with a Kd of about 7 microM. Thus, we suggest that a putative amphipathic alpha-helix between residues 52 and 69 of saposin B plays a major role in the recognition and binding of GM1-ganglioside by saposin B.