The regulation of membrane lipid biogenesis was investigated by measuring the levels of the acyl-acyl carrier protein (acyl-ACP) intermediates in the biosynthetic pathway. In particular, the role of the sn-glycerol-3-phosphate acyltransferase was assessed by focusing on the size and composition of the long chain acyl-ACP pool. The ACP( pool was specifically labeled in vivo with beta-[3-3H]alanine and the ACP subspecies analyzed by reversed phase liquid chromatography and conformationally sensitive gel electrophoresis. The acyl-ACP pool was found to be a small fraction of the total ACP in normally growing cells and was particularly devoid of chain lengths that could serve as acyltransferase substrates. Inhibition of phospholipid synthesis at the acyltransferase step resulted in a rapid increase in the content of acyl-ACP, and analysis showed the presence of chain lengths that are acyltransferase substrates. Acyl-CoAs were not detected during interruption of acyl transfer activity. These results show that 1) acyl-ACPs are the acyl donors for phospholipid synthesis in vivo, 2) the acyltransferase does not play a role in the regulation of the lipid biosynthetic rate or the composition of phospholipid acyl moieties, 3) the primary regulatory site in phospholipid biosynthesis is at an early step in fatty acid biosynthesis, 4) feedback regulation by long chain acyl-ACP's is not a controlling mechanism for fatty acid synthesis under normal physiological circumstances, and 5) enzymes that utilize acyl-ACPs are involved in kinetic competition for the scarce acyl-ACP substrates.