The hematoside of rat intestine is analyzed from 1 day to 60 days of age. During the first 3 weeks of life, GM3 (N-acetylneuraminylgalactosylglucosylceramide) contains only nonhydroxylated fatty acids and accounts for 80-90% of the ganglioside sialic acid. Its concentration is maximum at 6 days (315 micrograms of NeuAc/g of intestine) and falls abruptly over the next 2 weeks. It reaches 45 micrograms of NeuAc/g of intestine at 60 days. Between 28 and 60 days, GM3 accounts for 72% of the total intestinal gangliosides. From 21 days on, structural modifications of GM3 are observed. N-Acetylneuraminic acid is replaced progressively by N-glycolylneuraminic acid and nonhydroxylated fatty acids are replaced by alpha-hydroxylated fatty acids. Both changes are interpreted as the result of hydroxylations of GM3 components which are triggered at the time of weaning. These hydroxylations take place chiefly in epithelial cells and to a much lesser extent in nonepithelial residue, as shown by the separate analysis of both compartments of rat intestine at 38 and 60 days. In epithelial cells, the highest percentage of alpha-hydroxylated fatty acids and of N-glycolylneuraminic acid is found at 60 days. In addition, 4 D-hydroxysphinganine is the major base of the GM3 of intestinal cells from birth to adulthood.