In the present experiments, selective quenching by trinitrophenyl groups as well as steady-state fluorescence polarization and differential polarized phase fluorescence techniques, using three different lipid soluble fluorophores, were used to directly examine the fluidity of the exofacial and cytofacial leaflets of rat small intestinal brush-border membranes. These studies revealed that the fluidity of the exofacial hemileaflet was greater than the cytofacial hemileaflet. Differences in the distribution of phosphatidylcholine and phosphatidylethanolamine, as assessed by phospholipase A2 treatment and trinitrophenylation of aminophospholipids, were, at least partially, responsible for the asymmetrical fluidity of the hemileaflets. Moreover, in vitro addition of benzyl alcohol (final concn 25 mM) preferentially fluidized the exofacial leaflet and concomitantly decreased leucine aminopeptidase activity but did not affect the activities of maltase, sucrase, alkaline phosphatase, or gamma-glutamyltranspeptidase. In vivo addition of the membrane-mobility agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanate] (A2C) (final concn 7.5 microM) preferentially fluidized the cytofacial leaflet and increased Na(+)-gradient-dependent D-glucose transport but not Na(+)-gradient-dependent L-leucine transport.