Photosynthetically active chloroplasts retaining high rates of fatty acid synthesis from [1-(14)C]acetate were purified from leaves of both 16:3 (Solanum nodiflorum, Chenopodium album) and 18:3 plants (Amaranthus lividus, Pisum sativum). A comparison of lipids into which newly synthesized fatty acids were incorporated revealed that, in 18:3 chloroplasts, enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD) were significantly less active than in 16:3 chloroplasts. In contrast, labeling rates of MGD from UDP-[(14)C]gal were similar for both types of chloroplasts.The composition and positional distribution of labeled fatty acids within the glycerides synthesized by isolated 16:3 and 18:3 chloroplasts were similar and in each case only a C18/C16 diacylglycerol backbone was synthesized. In nodiflorum chloroplasts, C18:1/C16:0 MGD assembled de novo was completely desaturated to the C18:3/C16:3 stage.Whereas newly synthesized C18/C18 MGD could not be detected in any of these chloroplasts if incubated with [(14)C]acetate after isolation, chloroplasts isolated from acetate-labeled leaves contained MGD with labeled C18 fatty acids at both sn-1 and sn-2 positions. Taken together, these results provide further evidence on an organellar level for the operation of pro- and eucaryotic pathways in the biosynthesis of MGD in different groups of plants.