In addition to acyl‐CoA, purified glycerol‐3‐phosphate acyltransferases from pea and spinach chloroplasts can also use acyl‐(acyl‐carrier protein), acyl‐ACP, as a substrate for glycerol 3‐phosphate acylation. The enzyme fractions showed absolute specificity for glycerol 3‐phosphate as acyl acceptor. Dihydroxyacetone phosphate was ineffective. Glycerol 3‐phosphate was almost exclusively acylated at the C‐1 position. If mixtures of palmitoyl‐ACP, stearoyl‐ACP and oleoyl‐ACP were offered, the oleoyl group was preferred. These data fully agree with previous experiments on these enzymes carried out with various acyl‐CoA thioesters. Kinetic data determined with different acyl‐ACPs as substrates are consistent with the observed fatty acid selectivity for the oleoyl group. Double labelling experiments with mixtures of oleoyl‐ACP and oleoyl‐CoA demonstrated a preference for ACP‐thioesters.Monoacylglycerol‐3‐phosphate acyltransferase, localized in the envelope of chloroplasts, can also utilize acyl‐ACP as substrate. Envelope fractions of spinach as well as of pea showed a high specificity for the palmitoyl group when ACP‐thioesters or CoA‐thioesters were offered and directed this acyl group to the C‐2 position of the glycerol backbone. Results from competition experiments with [14C]palmitoyl‐ACP and [3H]palmitoyl‐CoA indicate that the membrane‐bound acyltransferase preferably uses ACP‐thioesters for the acylation of 1‐acylglycerol 3‐phosphate. According to fatty acid selectivities and specificities the main product of the recombined acyltransferase systems in chloroplasts of 16:3 plants as well as 18:3 plants is a phosphatidic acid with the oleoyl group at C‐1 and the palmitoyl group at C‐2 whereas molecules with the oleoyl group at both positions are not synthesized.Under appropriate conditions the phosphatidic acid formed by the soluble acyltransferase and spinach envelope was rapidly converted to monogalactosyl diacylglycerol in the presence of UDP‐galactose. In analogous assays with acyltransferase and envelope from pea only a low proportion of labelled phosphatidic acid was converted via diacylglycerol to monogalactosyl diacylglycerol. In both systems monogalactosyl diacylglycerol synthesized in the presence of C16:0‐thioesters and C18:1‐thioesters carried C18:1 at C‐1 and C16:0 at C‐2 in agreement with the fatty acid selectivity of the acyltransferase systems.