A gentle procedure allowed the isolation of intact and highly active chloroplasts from the unicellular green alga Acetabularia mediterranea. These chloroplasts incorporated carbon from NaH14CO3 into fatty acids and prenyl lipids at a rate of about 20-50 nmol carbon· (mg chlorophyll)-1·h-1. Most of the fatty acids formed in vitro were esterified in galactolipids. The main prenyl lipids synthesized were the chlorophyll side chain, intermediates of the carotenogenic path, α-and β-carotene, as well as lutein. Large amounts of [1-14C]acetate were incorporated, but exclusively into fatty acids. Isopentenyl diphosphate was a good substrate for prenyl-lipid formation in hypotonically treated chloroplasts. The envelope of intact chloroplasts, however, was impermeable to this compound. Intermediates of the mevalonate pathway were not accepted as precursors under conditions where isopentenyl diphosphate was well incorporated. The results show that the lipid biosynthetic pathways in the plastids of Acetabularia, a member of the ancient family of Dasycladaceae, are very similar to those in higher-plant plastids. © 1990 Springer-Verlag.