The alphavirus Semliki Forest virus (SFV) enters cells through receptor-mediated endocytosis. Subsequently, triggered by the acid pH in endosomes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presence of cholesterol in the target membrane. Recently, we have demonstrated that fusion of SFV also requires sphingolipids [Nieva, J. L., Bron, R., Corver, J., & Wilschut, J. (1994) EMBO J. 13, 2797-2804]. In the present paper, we show that the activation of low-pH-dependent fusion of SFV by sphingolipids is a stereospecific process. Pyrene-labeled SFV fused rapidly and extensively with liposomes consisting of a mixture of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, supplemented with low concentrations of D-erythro-ceramide, representing the naturally occurring sphingolipid stereoisomer. Fusion was assessed by a decrease in the pyrene excimer fluorescence. L-erythro-, D-threo-, and L-threo-ceramide did not support fusion of the virus. Similar results were obtained with the corresponding sphingomyelin stereoisomers. The stereospecificity of SFV fusion activation was confirmed by using an assay based on degradation of the viral capsid protein by trypsin encapsulated in the target liposomes. Fusion mediated by D-erythro-ceramide was not affected by the additional presence in the target liposomes of ceramide stereoisomers incapable of fusion activation. Binding of the virus to the liposomes, as assessed by flotation on sucrose density gradients, was not dependent on the presence of fusion-competent or fusion-incompetent sphingolipids in the liposomes. The results of this study support the notion that a stereospecific interaction of the viral fusion protein with D-erythro sphingolipids in the target membrane represents an essential step in the activation of the fusion capacity of SFV.