Characteristics of small intestinal microvillus membrane lipid composition and associated lipid fluidity were studied throughout postnatal development in the rabbit. Microvillus membranes from jejunal and ileal epithelia were isolated in high purity from 14 days to adulthood. Significant jejunal-ileal differences in microvillus membrane cholesterol to phospholipid molar ratios were observed at all ages. Despite a relatively constant cholesterol content (as percentage of total lipids) both regionally and at all ages, marked increases in the microvillus membrane cholesterol to phospholipid ratio were observed after weaning in both jejunum (14 days = 0.56, adult = 0.83) and ileum (14 days = 0.66, adult = 0.98). Further age-related changes in microvillus membrane lipid composition were characterized by a significant reduction in the weight ratio of saturated to unsaturated fatty acids in jejunum and ileum and by the emergence, in the mature animal, of a jejunal-ileal difference in total lipid content (jejunum = 1.21 mg/mg protein, ileum = 1.01 mg/mg protein). Changes in microvillus membrane lipid fluidity estimated by fluorescence polarization spectrometry directly paralleled changes in the cholesterol to phospholipid molar ratio. Fluorescence anisotropy of jejunal and ileal microvillus membranes increased during the weaning period, suggesting decreased membrane fluidity. At all ages, ileal membranes were relatively less fluid than jejunal microvillus membrane. Studies of protein-free microvillus membrane liposomes, prepared from extracted membrane lipid, confirmed a major lipid-dependent thermotropic transition point at approximately 23 degrees C in adult jejunal and approximately 25 degrees C in adult ileal enterocyte membranes, although no clear transition point was observed in suckling microvillus membrane. These combined data suggested that significant alterations in the microvillus membrane lipid composition and its physical-chemical properties occur during postnatal development in both jejunal and ileal rabbit small intestinal microvillus membrane. It is proposed that membrane lipid-dependent structural characteristics may be directly related to the maturation of enterocyte transport function.