The normal biophysical properties of the maturing mammalian enterocyte membrane are poorly understood. While the effects of glucocorticoids on maturation of intestinal enzyme function has been intensively investigated, their effects on membrane biophysical properties are not known. We used 1,6-diphenyl-1,3,5-hexatriene as a probe in fluorescence anisotropy studies to determine the fluidity of rat brush border membrane. Maturational changes and the effects of glucocorticoids administered antenatally or postnatally were determined. Fluorescence anisotropy values for 1,6-diphenyl-1,3,5-hexatriene in mature brush border membranes were higher than those values in membranes obtained from younger animals reflecting a less fluid membrane. Glucocorticoids administered to suckling rats increased the anisotropy values of 1,6-diphenyl-1,3,5-hexatriene in the membranes compared to saline-administered littermates. The anisotropy of intestinal brush border membrane was also increased in fetal rats whose mothers received dexamethasone. These alterations may relate to protein-binding properties and permeability characteristics of the enterocyte membrane.