We examined biotin transport across the basolateral membrane (BLM) of rat liver using BLM vesicles (BLMV) technique. The purity and suitability for transport studies of liver BLMV were demonstrated by morphological (electron microscopy), enzymatic, and functional criteria. Orientation of liver BLMV was determined by freeze-fracture electron microscopy and by [3H]ouabain binding methodology and was found to be 65.3-69.7% in the right-side-out orientation. Uptake of biotin by liver BLMV was found by osmolarity and temperature studies to be mostly the result of transport of the substrate into an active intravesicular space with little binding to membrane surfaces. Transport of biotin was found to be Na+ gradient dependent with a distinct "over-shoot" phenomenon. Initial rate of transport of biotin as a function of concentration was found to include a saturable component in the presence of a Na+ gradient (out greater than in) but was linear and lower in the presence of a K+ gradient (no Na+). Kinetic parameters of the saturable Na+ gradient-dependent transport process were 0.39 microM and 1,807 fmol.mg protein-1.20 s-1 for the apparent Km and Vmax, respectively. In the presence, but not the absence, of a Na+ gradient (out greater than in), the addition of structural analogues to the incubation medium caused significant inhibition in the transport of 0.079 microM [3H]biotin. Induction, with the use of valinomycin and an inwardly directed K+ gradient, of a relatively positive intravesicular space caused significant inhibition in the initial rate of biotin transport.(ABSTRACT TRUNCATED AT 250 WORDS)