Isolated intestinal mucosa cells of rats were used to investigate the intestinal transport of biotin. This method utilizing a double-label isotope technique showed that uptake could not be saturated, even in a wide range of biotin concentrations (0.01-2 microM). A metabolic inhibitor (antimycin A) did not prevent cell uptake of biotin. The transport mechanism was independent of temperature (Q10 = 1.04). When excess biotin was added to the incubation medium, there was no efflux of the vitamin from intestinal cells. The results also showed that the cells did not concentrate the vitamin, regardless of its concentration in the incubation medium. The mechanism of biotin uptake by rat cells at physiological concentrations is thus a passive diffusion phenomenon.