We examined the relationship between reduced galactolipid content in tomato fruit at 4C and chilling injury. Galactolipid biosynthesis from 14C-acetate was compared in pericarp discs of cold-tolerant `New York 280' (`NY') and -sensitive `Early Cherry' (`EC') at 4C and 20C. Labeled lipids were separated by 2D-TLC. Labeled monogalactosyldiglyceride (MGDG) molecular species were hydrolyzed using a position-specific lipase; the fatty acids released were hydrogenated and separated according to chain length by reverse-phase TLC. At 4C, the relative amount of radioactivity was reduced in MGDG and enhanced in phosphatidylcholine (PC) in both cultivars, in comparison with labeling at 20C. In discs from fruit chilled for 6 h, labeling was similar in `NY' and `EC'. In fruit held at 4C for 8 days, labeling of MGDG was reduced and that in PC was enhanced to a greater extent in chilling-sensitive `EC' than in `NY'. The proportion of the MGDG label in eukaryotic species (i.e., the ratio in C18/C16 fatty acids in position sn-2), was less in `EC' at 4C than at 20C, even for fruit held at 4C for only 6 h. The ratio was little affected in `NY'. The data indicate that biosynthesis of eukaryotic MGDG was inhibited in tomato fruit at chilling injury-inducing temperatures.