The lipids of enveloped viruses play critical roles in viral morphogenesis and infectivity. They are derived from the host membranes from which virus budding occurs, but the precise lipid composition has not been determined for any virus. Employing mass spectrometry, this study provides a quantitative analysis of the lipid constituents of HIV and a comprehensive comparison with its host membranes. Both a substantial enrichment of the unusual sphingolipid dihydrosphingomyelin and a loss of viral infectivity upon inhibition of sphingolipid biosynthesis in host cells are reported, establishing a critical role for this lipid class in the HIV replication cycle. Intriguingly, the overall lipid composition of native HIV membranes resembles detergent-resistant membrane microdomains and is strikingly different from that of host cell membranes. With this composition, the HIV lipidome provides strong evidence for the existence of lipid rafts in living cells.