Two different enzymes for galactosylation occur in isolated chloroplast envelopes of spinach leaves, UDPgalactose-diglyceride galactosyltransferase and galactolipid-galactolipid galactosyltransferase. The first enzyme is responsible for the biosynthesis of monogalactosyldiglyceride, UDPgalactose being donor of the galactosyl moiety. The second enzyme is responsible for the biosynthesis of digalactosyldiglyceride and higher homologues. The optimum pH for monogalactosyldiglyceride synthesis was found to be 7.5, for digalactosyldiglyceride synthesis 6.5. After incubation at pH 7.4 a Lineweaver-Burk plot indicated two binding sites for UDPgalactose on the UDPgalactose-diglyceride galactosyltransferase with different affinities for UDPgalactose and different activities. Indirectly the galactolipid-galactolipid galactosyltransferase was shown to respond similarly to various UDPgalactose concentrations. However, the second reaction also proceeds in absence of UDPgalactose. It was concluded that the second enzyme does not require the presence of UDPgalactose, but that galactosyl transfer proceeds by direct exchange of galactosyl groups between molecules of galactolipids, or via unknown lipid intermediates, not detected in our system. Results of other investigations will be discussed in the light of the present data.