The objectives of this study were to determine mRNA expression of monocarboxylate transporters (MCT) and to evaluate intestinal transport of the MCT substrates gamma-hydroxybutyrate (GHB) and d-lactate in human intestinal Caco-2 cells. The presence of mRNA for MCT1, 2, 3, and 4 was observed in Caco-2 cells. The uptake of both GHB and d-lactate in Caco-2 cells was demonstrated to be pH- and concentration-dependent and sodium-independent. The uptake of GHB and d-lactate was best described by a Michaelis-Menten equation with passive diffusion (GHB: K(m) = 17.6 +/- 10.5 mM, V(max) = 17.3 +/- 11.7 nmol/min/mg, and P = 0.38 +/- 0.15 microl/min/mg; and d-lactate: K(m) = 6.0 +/- 2.9 mM, V(max) = 35.0 +/- 18.4 nmol/min/mg, and P = 1.3 +/- 0.6 microl/min/mg). The uptake of GHB and d-lactate was significantly decreased by the known MCT inhibitor alpha-cyano-4-hydroxycinnamate and the MCT substrates GHB and d-lactate but not by the organic cation tetraethylammonium chloride. Directional flux studies with both GHB and d-lactate suggested the involvement of carrier-mediated transport with the permeability in the apical to basolateral direction higher than that in the basolateral to apical direction. These findings confirm the presence of MCT1-4 in Caco-2 cells and demonstrate GHB and d-lactate transport characteristics consistent with proton-dependent MCT-mediated transport.