The effect of tryptophan on sodium and chloride fluxes was determined simultaneously on paired flat-sheet preparations of newborn rabbit jejunum under short-circuit conditions. In the absence of amino acids, the newborn rabbit jejunum actively absorbed sodium, and possibly bicarbonate, whereas chloride appeared to be distributed passively across the jejunum. Tryptophan (2 mM) caused an increment in short-circuit current (Isc) that was due to an increase in net active sodium flux and had no significant effect on tissue conductance. At a 10 mM concentration, tryptophan initially increased Isc, although not to as large a degree as phenylalanine or alanine, and then caused a progressive decline that reached a plateau around 60 min. The reduction in Isc was attributed primarily to abolition of sodium absorption and stimulation of chloride secretion. Tryptophan reduced the unidirectional fluxes of sodium, increased those of chloride, and decreased total tissue conductance. These results suggest that 10 mM tryptophan initially causes a predicted increase in sodium absorption, followed by an inhibition of sodium absorption and stimulation of chloride secretion. The mechanism of action of tryptophan resulting in changes in ion fluxes is unknown.