The mechanism of riboflavin (RF) uptake by intestinal basolateral membrane vesicles (BLMV) was examined in this study. BLMV were isolated by an established Percoll-gradient methodology from rabbit small intestine. Uptake of riboflavin was mainly the result of transport of the substrate into an osmotically active intravesicular space with less binding to membrane surfaces. Uptake of RF with time was similar in the presence of a Na+ and a K+ gradient (out > in) and was not significantly influenced by changes in incubation buffer pH. The initial rate of uptake of riboflavin as a function of concentration was saturable in both jejunal and ileal BLMV and occurred with apparent Km values of 5.0 microM and 4.4 microM and Vmax values of 91.6 and 60.8 pmol/mg protein per 5 s, respectively. Unlabeled riboflavin and the structural analogues lumiflavin, isoriboflavin and 8-aminoriboflavin all caused significant inhibition (but to different degrees) in the uptake of [3H]riboflavin. On the other hand, 8-hydroxyriboflavin, lumichrome, lumazine and D-ribose failed to inhibit [3H]riboflavin uptake. Trans-stimulation of [3H]riboflavin efflux from preloaded BLMV by unlabeled riboflavin or lumiflavin was also observed. Altering transmembrane electrical potential by anion substitution and valinomycin-induced K+ diffusion did not affect the riboflavin uptake process. These results demonstrate the existence of a specialized carrier-mediated mechanism for riboflavin uptake by intestinal BLMV. Furthermore, the system appears to transport the vitamin by a process which is Na(+)- and pH-independent, and electroneutral in nature.