Abstract: The proton pumping [H+]-ATPase, Pma1p, is an abundant and very long-lived polytopic protein of the S. cerevisiae plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as an excellent model to study plasma membrane biogenesis. We have previously shown that newly synthesized Pma1p is mistargeted to the vacuole in an elo3 mutant that affects the synthesis of the ceramide-bound C26 very long-chain fatty acid (Eisenkolb et al. (2002). Mol. Biol. Cell 13, 4414-4428) and now describe a more detailed analysis of the role of lipids in Pma1p biogenesis. Remarkably, a block at various steps of sterol biosynthesis, a complete block in sterol synthesis, or the exchange of internally synthesized ergosterol by externally supplied ergosterol or even by cholesterol does not affect Pma1p biogenesis, or its association with detergent resistant membrane domains (lipid "rafts"). However, a block in sphingolipid synthesis or any perturbation in the synthesis of the ceramide-bound C26 very long-chain fatty acid, results in mistargeting of newly synthesized Pma1p to the vacuole. Mistargeting correlates with a lack of newly synthesized Pma1p to acquire detergent resistance, suggesting that sphingolipids with very long acyl chains affect sorting of Pma1p to the cell surface.