The lack of an in vivo method for measuring influx of amino acid into the mucosa has prevented a systematic comparison of characteristics of amino acid influx in vivo with prior in vitro studies. We developed and validated a technique for measuring amino acid influx in vivo. The mucosa is exposed briefly to labelled amino acid perfused luminally at a rapid rate and tissue uptake is measured. The brief exposure period insures that amino acid is confined to the segment. The rapid perfusion rate minimizes concentration of endogenous Na+ in the lumen and permits Na-dependency for alpha-aminoisobutyric acid influx to be demonstrated in vivo for the first time. We also demonstrated the inhibitory effect of K+ and competition by glycine on alpha-aminoisobutyric acid influx in vivo. The saturation kinetics for L-leucine in vivo and in vitro were compared under varying perfusion rates and with and without stirring with air. Under optimal conditions of agitation (rapid perfusion and bubbling with air), the apparent Michaelis constant (Kt) is decreased to be almost equal to that determined under comparable influx conditions in vitro. These studies demonstrate no major difference between characteristics of amino acid transport under more physiologic in vivo conditions as compared with prior in vitro studies.