Intensive interest is currently focused on the role of ceramide (1), a key lipid molecule that functions as a second messenger. The first asymmetric synthesis of a D-erythro-ceramide analogue that contains a C(5)-C(6) trans double bond (3), together with its biological evaluation in a viral-liposome fusion system, is described. Sharpless asymmetric dihydroxylation of 4'-methoxyphenyl trans-5-octadecyn-2-enyl ether (enyne 8a), prepared by the coupling reaction of 1-[(E)-(4'-bromo-2'-butenyl)oxy]-4-methoxybenzene (7) with Lithium tetradecyne, generated yne-diol 9 in 96% yield with the desired stereochemistry at the C(2)and (3) positions and high enantiomeric purity (95% enantiomeric excess). Birch reduction (Li/EtNH2) of yne-diol 9 furnished (2R,3R,5E)-octadecene-1,2,3-triol (10) stereospecifically. The latter was converted to 2 azido derivative 13 in three steps (via a 2-O-triflate-1,3-O-benzylidene intermediate) and 55% overall yield. Reduction of azide 13 and in situ N-acylation with p-nitrophenyl cis-hexadec-4-enoate provided D-erythro-Delta(5)-trans-ceramide (3) in 91% yield. The role of the trans double bond of ceramide in mediating fusion of an alphavirus (Semliki Forest virus) was assessed in a liposomal model system, using target phospholipid/cholesterol vesicles containing either D-erythro-ceramide 1 or 3. The kinetics of virus fusion, as monitored by a change in pyrene excimer fluorescence over a period of 60 s, showed that Delta(5)-trans-ceramide 3 was completely inactive, indicating that there is an absolute requirement for the trans double bond to be located between C(4) and C(5). These data indicate that the molecular determinants on the viral envelope glycoprotein are highly specific for recognition of the unsaturated site in the ceramide molecule.