A fatty acid desaturase mutant was used to study the regulatory effects of unsaturated fatty acids on glycerolipid biosynthesis in yeast. Cells grown on palmitoleic acid (16:1) contain approximately twice the amount of phospholipids and triacylglycerols (per mg. dry weight) compared to those grown on oleic acid (18:1). The in vitro specific activity of glycerol-3-phosphate acyl transferase was two fold higher when palmitoyl-CoA was used as a substrate relative to oleoyl-CoA. In vivo methylation studies revealed that cells grown on palmitoleic acid produce 2.6 fold more phosphatidylcholine via the CDP-DAG (methylation) pathway than cells grown on oleic acid, although oleic acid facilitated the direct phosphorylation of exogenously supplied choline. These data indicate that unsaturated fatty acids may act as key regulatory molecules which influence the glycerolipid biosynthetic matrix in yeast.