Acyl carrier protein (ACP coli) was isolated from commercially grown Escherichia coli B and was acetylated by chemical methods. Biological activity of the synthesized acetyl-ACP coli was checked in an in vitro fatty acid-synthesizing system isolated from E. coli B. Since acetyl-ACP is preferred over acetyl-coenzyme A (CoA) as a substrate in these reactions, the possibility that it may substitute for acetyl-CoA in biosynthetically and oxidatively important cellular pathways (glyoxylate and Krebs cycles, respectively) was examined. Acetyl-ACP was tested for substrate activity with the enzyme of each cycle which has been found to utilize acetyl-CoA. Crystalline citrate synthase (EC 4.1.3.7) of porcine origin (Calbiochem) was found to be inactive with acetyl-ACP coli, which acted neither as a substrate nor as an inhibitor in the presence of acetyl-CoA. Malate synthase (EC 4.1.3.2) of the acetate type was isolated from acetate-grown cells of a mutant of E. coli K-12 (VGD(3)H(5)) and was also found to be inactive with acetyl-ACP coli. The significance of these results and of the recent discovery of another phospho-pantetheine-containing protein are discussed.