The uptake of spermine by isolated rat intestinal brush-border membrane vesicles was studied. Uptake was biphasic, with an initial rapid uptake followed by a prolonged slower phase. Spermine uptake was not affected by a Na+ electrochemical gradient. The equilibrium uptake of spermine was considerably dependent upon the medium pH. At pH 7.5 the degree of uptake was higher than that at pH 6.5 and was inversely proportional to the extravesicular osmolarity with a relatively high binding, which was estimated by extraporation to infinite extravesicular osmolarity (zero intravesicular space), while the uptake at pH 6.5 was not altered under the various medium osmolarities. A kinetic analysis of the initial uptake rate of spermine at 37 degrees C gave a Km of 24.2 microM and Vmax of 206.1 pmol/mg protein per min. Furthermore, the uptake at 4 degrees C was nonlinear, providing evidence for saturability. These findings suggest that spermine was associated with intestinal brush-border membrane vesicles in two ways, by binding to the outside and inside of membrane vesicles. The interaction of spermine and the apical membrane can be a contributory factor in the accumulation of this polyamine in the intestine of the intact animal.