Effects of MgCl(2) and free fatty acids (FFA) on galactolipid:galactolipid galactosyltransferase (GGGT) and UDP-galactose: 1,2-diacylglycerol galactosyltransferase (UDGT) in chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.) leaves were examined. GGGT activity was sigmoidally stimulated by MgCl(2) with a saturated concentration of more than 5 millimolar. Free alpha-linolenic acid (18:3) caused a drastic increase in GGGT activity under limiting concentrations of MgCl(2), without affecting its maximum activity at higher MgCl(2) concentrations. Free 18:3 alone did not affect the GGGT activity. The effective species of FFA for the stimulation of GGGT activity in the presence of MgCl(2) were unsaturated 16- and 18-carbon fatty acids. GGGT activity was also stimulated by 18:3 in the presence of MnCl(2), CaCl(2) and a high concentration of KCl in place of MgCl(2). UDGT activity was hyperbolically enhanced by MgCl(2) with a saturated concentration of 1 to 2 millimolar. In contrast to GGGT, UDGT was severely inhibited by 18:3, and MgCl(2)-induced stimulation was completely abolished by 18:3. Unsaturated 16- and 18-carbon fatty acids were more inhibitory to UDGT than the saturated acids. The dependence of GGGT activity on monogalactosyldiacylglycerol (MGDG) and MgCl(2) concentrations was identical in the envelope membranes isolated from non- and ozone (0.5 microliter/liter)-fumigated spinach leaves, indicating that GGGT remained active in the leaves during ozone fumigation. The results are discussed in relation to the regulation of galactolipid biosynthesis by the endogenous FFA in the envelopes and to the involvement of GGGT in the triacylglycerol synthesis from MGDG in ozone-fumigated leaves.