Vectorial (re)absorption of amino acids across epithelial barriers of the small intestine and the kidney proximal tubule requires that amino acids with various properties be transported against their concentration gradients across two cellular membranes. After having isolated and characterized in the oocyte expression system three epithelial heterodimeric amino acid exchangers expressed in transporting epithelia, we are now investigating their role and functional co-operation in vectorial transport. Using MDCK cells for exogenous expression, we show within an epithelium that the luminal uptake of L-cystine and cationic amino acids depends on the formation of a heterodimer of the glycoprotein rBAT and the multitransmembrane protein b^0,+AT. Expression of y⁺LAT1 and 4F2hc in the MDCK epithelium expressing rBAT-b^0,+AT promotes transepithelial exchange of apical cationic amino acids against basolateral neutral amino acids, demonstrating the functional co-operation of apical rBAT-b^0,+AT with basolateral 4F2hc-y⁺LAT1, for cationic amino acid (re)absorption. The additional expression of LAT2 provides a basolateral exchanger for neutral amino acids that leads to an increase in basolateral-to-apical transport of neutral amino acids. This inverse transport (basolateral-apical) of neutral amino acids indicates that MDCK epithelia lack unidirectional pathways that recycle the neutral amino acids exchanged against the cationic ones. To investigate the export function of 4F2hc-LAT2 and that of the related ubiquitous 4F2hc-LAT1 complex, we have used the oocyte expression system and measured the efflux of (injected) amino acids (tracer studies and measurements by HPLC) as well as the transtimulation of influx by pre-injected amino acids. This study shows a clear difference in intracellular substrate specificity between the two LAT transporters and a transporter-specific asymmetry in the handling of some substrate amino acids. In summary, our work illustrates the role of exchangers for transepithelial transport of some substrates and shows that, in the absence of adequate parallel unidirectional transporters, some exchanged substrates are transported in the inverse direction. Thus co-operation of unidirectional transporters with exchangers is required to drive the vectorial transport of all amino acids across an epithelium.