Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acyl-CoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V(max) of 8.7 nmol/min/mg protein and an apparent K(m) of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn(2+) ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [(3)H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked. This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.