Nitrogen load can be reduced by substituting dietary protein intake with keto or hydroxy analogues of amino acids. To investigate their intestinal absorption, human jejunal brush border membrane vesicles were used to measure uptake of L-leucine hydroxy analogue (L-LHA) and L-lactate. Uptake assays were performed under voltage-clamped conditions in the presence of valinomycin and K+ inside and outside. Both inward directed H(+)- and Na(+)-gradients stimulated uptake of both substrates. The H(+)-gradient was the major driving force and led to an increased rate of L-LHA or L-lactate transport. The proton ionophore FCCP abolished the H(+)-gradient-driven but not the Na(+)-gradient-driven uptakes of both substrates. The H(+)-gradient-driven uptake of both substrates was trans-stimulated by L-LHA, D-LHA, L-valine hydroxy analogue or L-lactate, respectively. In the presence of a Na(+)-gradient the uptake of tracer L-lactate was trans-stimulated only after preloading the vesicles with L-lactate but not by L-LHA. It can be concluded that the H(+)-gradient-driven transport of L- or D-hydroxy analogues of branched chain amino acids and L-lactate across the human intestinal brush border membrane is mediated by the same carrier.