Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-(14)C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-(14)C]acetate or sn-[U-(14)C]glycerol-3-P in the presence of Mg(2+) and unlabeled UDP-Gal resulted in high (14)C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.