The lipid monogalactosyl diacylglycerol (MGD) is a major structural component of photosynthetic membranes in chloroplasts. Its formation is catalyzed by the enzyme MGD synthase. In many plants, MGD derives from two different biosynthetic pathways: the prokaryotic pathway, which operates entirely within the plastid, and the eukaryotic pathway, which involves steps in the endoplasmic reticulum. Here, we describe the identification and characterization of an Arabidopsis mutant with a defective MGD synthase gene (MGD1). The mutant was identified in a screen of T-DNA lines for individuals with defects in chloroplast biogenesis. It has a yellow-green phenotype that correlates with a approximately 50% deficiency in total chlorophyll per plant. A single T-DNA insertion is located adjacent to the transcription initiation site of the MGD1 gene, and the abundance of MGD1 mRNA is reduced by 75% compared with wild type. Correlation between steady-state MGD1 transcript levels and MGD synthase activity (also reduced by 75% in mgd1) suggests that MGD1 is the most important MGD synthase in green tissues. The amount of MGD in mutant leaves is reduced by 42% compared with wild type. MGD from the mutant contains 23% less 16:3 fatty acid and 10% more 18:3 fatty acid. Because 16:3 is a characteristic feature of MGD from the prokaryotic pathway, it is possible that MGD1 operates with some preference in the prokaryotic pathway. Finally, the MGD-deficiency of mgd1 is correlated with striking defects in chloroplast ultrastructure, strongly suggesting a unique role for MGD in the structural organization of plastidic membranes.