The current research investigated the regulation of monogalactosyldiacylglycerol (MGDG) biosynthesis, catalyzed by MGDG synthase (MGD) (UDP-galactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46), during chloroplast development in cucumbers (Cucumis sativus L. cv. Aonagajibai). In etiolated seedlings, white light induced a transient increase in MGD mRNA, followed by a subsequent increase in enzyme activity. MGDG, digalactosyldiacylglycerol (DGDG), and linolenic acid (18 : 3) of both MGDG and DGDG accumulated in a light-dependent manner. Early light-dependent induction of MGD protein was also identified in isolated chloroplasts. When cotyledons were detached from seedlings, these light-induced changes diminished. However, when a synthetic cytokinin, benzyladenine, was added to the detached cotyledons, a transient increase in MGD mRNA and a linear increase in the enzyme activity were induced even in the dark. Galactolipids subsequently accumulated to some extent and 18 : 3 content also increased. MGDG fully accumulated in detached cotyledons with co-treatment of light and a cytokinin. Red light (>600 nm) and far-red light (>700 nm) both induced an increase in MGD mRNA and enzyme activity but far-red light did not induce an accumulation of MGDG. These results suggest that (1). galactolipid biosynthesis is regulated by the cooperation of light and a cytokinin; (2). the accumulation of MGDG requires cytokinin in addition to light; (3). a red light (600-700 nm) dependent factor is necessary for the maximal galactolipid accumulation in addition to increase in MGD transcript and activity.