Seedlings of Arabidopsis thaliana (L.) Heynh. fail to become green when germinated and grown on media containing high concentrations of glucose (Glc). Although previous studies have shown that sugar concentration affects chlorophyll levels and photosynthetic gene expression, the possibility that sugar concentration might affect actual chloroplast biogenesis has received little attention. Therefore, experiments were conducted to determine whether germination and growth on Glc impairs development of mature chloroplasts from the proplastids found in plant embryos. To monitor chloroplast biogenesis, the levels of a chloroplast-specific fatty acid, hexadecatrienoic (16:3) fatty acid, were measured in Arabidopsis seedlings grown on media containing different concentrations of Glc. These experiments indicate that moderate concentrations of Glc delay accumulation of 16:3. The effects of Glc on 16:3 levels are not solely due to osmotic stress, as equi-molar and even twice equi-molar concentrations of sorbitol do not exert comparable effects. Seedlings grown on concentrations of Glc high enough to prevent greening accumulate almost no 16:3, even after 22days of growth under continuous light conditions. The lack of 16:3, a major structural component of chloroplast membranes, suggests that seedlings do not develop mature chloroplasts when grown in the presence of high concentrations of exogenous Glc. Further support for this hypothesis is provided by electron microscopy studies revealing that seedlings grown on high concentrations of Glc lack identifiable chloroplasts. Although Glc has been reported to inhibit chloroplast development in unicellular organisms, similar studies on intact higher plants have been lacking.