Simple diffusion has been reported as the mechanism of biotin transport in rabbit intestine. In this study, we reevaluated this concept by examining biotin transport in rabbit intestine using optimal experimental conditions and a well-established brush-border membrane vesicles (BBMV) technique. Uptake of biotin by rabbit intestinal BBMV was found by an osmolarity study to be mostly the result of transport of the vitamin into an osmotically sensitive intravesicular space with little binding to membrane surfaces. Biotin transport in rabbit intestinal BBMV was 1) Na+ gradient dependent (out greater than in) with a clear "overshoot" phenomenon, indicating the accumulation of the substrate against a concentration gradient; 2) initial rate of biotin transport by the Na+ gradient-dependent component was saturable as a function of substrate concentration with apparent Km and maximum velocity (Vmax) values of 6.7 microM and 10.7 pmol.mg protein-1 x 10 s-1, respectively; 3) inhibited by high concentrations of unlabeled biotin and its related compounds desthiobiotin and thioctic acid in the presence, but not absence, of a Na+ gradient; and 4) not affected by inducing a relatively positive or negative intravesicular space with the use of valinomycin-induced K+ diffusion potential. These findings indicate that the biotin transport mechanism in rabbit intestine is carrier mediated in nature. Furthermore, this mechanism is Na+ gradient dependent, capable of accumulating the substrate against a concentration gradient and transport the vitamin via an electroneutral process.