The absorption of selected water-soluble vitamins was studied in isolated intestinal sleeves of channel catfish (Ictalurus punctatus). The uptakes of riboflavin and biotin were each inhibited by their nonlabeled form, indicating the presence of a saturable mechanism [riboflavin: Michaelis constant (Km), 4.84 +/- 1.23 microM; maximal velocity (Vmax), 0.59 +/- 0.07 pmol.mg-1.min-1 biotin: Km, 22.3 +/- 6.95 microM; Vmax, 0.67 +/- 0.10 pmol.mg-1.min-1]. In contrast, the uptakes of nicotinamide, folic acid, and its metabolic derivative 5-methyltetrahydrofolate were not significantly inhibited by their respective nonlabeled forms. Their uptakes were a linear function of concentration [dissociation constant, 0.173 +/- 0.006, 0.050 +/- 0.002, and 0.031 +/- 0.004 pmol.mg-1.min-1.microM-1, respectively]. Folic acid was absorbed more rapidly than 5-methyltetrahydrofolate, and neither vitamin inhibited the absorption of the other. Intestinal uptake of riboflavin and biotin is carrier mediated, whereas uptake of nicotinamide, folic acid, and 5-methyltetrahydrofolate occurs by simple diffusion. These mechanisms are similar to those found in mammals for the same vitamins, except for the hydrophobic folates, which are actively transported in mammals but where diffusion may suffice to fulfill metabolic requirements of catfish.