The G3P level in chloroplasts, rapidly isolated from spinach leaves during a light-dark cycle, oscillated between 2.5 and 4 nmol · mg -1 Chi, which corresponds to a concentration of nearly 0.1 -0.2 mᴍ. In order to study the role of the stromal G3P level on chloroplast lipid biosynthesis, G3P uptake, measured by silicone oil centrifugation, has been correlated with the lipid synthesizing capacity of intact spinach chloroplasts. The level of G3P in the chloroplast stroma was decreased by high orthophosphate (P i ) concentrations in the medium. This decrease was caused by a strong P i transport into the stroma, which is counterbalanced by a release of phosphorylated metabolites including G3P, mediated by the translocator. But because the reduced stromal G3P concentration exceeded about 3 times that for half saturation of the primary G3P acylation with oleoyl-ACP as preferred fatty acid donor, glycerolipid synthesis was not eliminated. Instead, the lowered G3P level in the stroma space limited the secondary acylation step, indicated by the reduced incorporation of palmitic acid into the diglyceride fraction, and led to an accumulation of free oleic acid. Thus, beside its function as primary acyl acceptor, the stromal G3P level apparently controls the pool size of ACP bound palmitic acid by limitation of the chain elongation step from palmitoyl- to stearoyl-ACP in order to induce the specific palmitic acid channeling into the C-2-position of chloroplast lipids by the secondary G3P acylation. A similar function may be due to fatty acid consuming reactions from outside the chloroplast like acyl-CoA thioester formation in the outer envelope membrane, stimulated by exogenous CoA and ATP. In contradiction to earlier findings intact Percoll chloroplasts showed a measurable glycerolipid labelling (3-4%) from exogenous [ 14 C]oleoyl-CoA in the presence of G3P (0.5 mᴍ), although most of the radioactivity was found in the free fatty acid fraction (7-10%). Incorporation into diglycerides was increased two-fold under fatty acid synthesizing conditions in the medium and the resulting diglycerides were further galactosylated by UDP- galactose addition. The latter observations suggest, that even in spite of the envelope impermeability for physiological concentrations of long-chain acyl-CoA thioesters, fatty acid transfer from these substrates to typical chloroplast lipids cannot be totally excluded.