The overall objective of this research is to study the mechanism of membrane biogenesis and membrane selectivity. Interest in the study of sphingolipids has been stimulated by recent observations relating these lipids to intracellular recognition, immunological determinants, and ion transport. The specific purpose of this project will be to determine the mechanism of synthesis of sphingosine, and the in vivo pathway for the biosynthesis of sphingomyelin and glucosylceramide. In vivo studies will be done with hamster cells grown in tissue culture. We will determine the in vivo occurrence of postulated intermediates in the tissue culture. We will determine the in vivo occurrence of postulated intermediates in the biosynthesis of spingosine such as: 3 keto-sphingosine and -dihydrosphingosine, 3 keto-sphingosine and -dihydrosphingosine containing-ceramides, -sphingomyelins and of phytosphingosine. For those intermediates that are detected an attempt will be made to develop a cell free system for their synthesis. We will also determine whether the in vivo pathway for the biosynthesis of sphingomyelin involves a condensation of CDP-choline with ceramides or sphingosylphosphoryl choline with a fatty acyl CoA derivative. We will examine whether the in vivo synthesis of glucosylceramide occurs via a condensation of UDP-glucose with ceramides or glucosylsphingosine with a fatty acyl CoA derivative. These studies will constitute the groundwork for further investigations on (1) subcellular localization of these enzymes, and (2) attempts to modify the sphingolipid composition of cells in culture, enabling one to correlate changes in composition with alterations in proposed functions ascribed to sphingolipids.