The effects of some amino acids,l-alanine,l-phenylalanine,dl-alanine,d-alanine and β-alanine on membrane potential, membrane current, amylase secretion and45Ca and86Rb fractional efflux in isolated mouse pancreatic segments were investigated. A two micro-electrode voltage clamp technique was applied to study the effects of the amino acids on membrane current. The amino acids evoked dose-dependent (0.05–0.5 mmole) and reversible membrane depolarization and increases in membrane current. The relative potencies of the actions of the amino acids were:l-alanine>dl-alanine>l-phenylalanine>d-alanine>β-alanine. A more detailed study of the action ofl-alanine showed that the relationship between thel-alanine-evoked membrane current and membrane potential was virtually linear with reversal of current polarity being observed at a membrane potential of about +30 mV. While thel-alanine-induced increase in membrane conductance was dose-dependent, the reversal potential (El-ala) was independent of thel-alanine concentration used. Replacement of the normal Na-rich superfusion fluid by a low Na solution (5 mM) markedly reduced thel-alanine-elicited inward current at the normal resting potential. Thel-alanine-evoked conductance increase was also reduced in low Na solution and the El-alawas close to 0 mV. During the exposure of pancreatic segments to Cl free solution (sulphate substitution) El-alawas about 12 mV more positive (+43.7±0.8 mV) than during exposure to control solution (+31.5±1.0 mV). Calculations based on the Goldman-Hodgkin-Katz equations taking into account the values of El-alain the different ionic situations, indicate that the relative permeability of the ionic pathway opened up in the presence ofl-alanine isPNa/PCl/PK=7.5/1.5/1.0 The pathway is therefore Na selective, but with some leak permeability for Cl and K. In the physiological range of membrane potentials (−15 to −40 mV) the current evoked byl-alanine is virtually entirely carried by Na. Measurements of amylase secretion and45Ca fractional efflux show that none of the amino acids (all at 10 mM concentrations) had significant effect whereas pentagastrin (Pn; 10−6M) elicited marked increases in amylase output and45Ca release. Both pentagastrin andl-alanine stimulated the release of86Rb from prelabelled tissue whereasdl-alanine,l-phenylalanine,d-alanine and β-alanine had little or no effect. The present results indicate that although the amino acids have no effect on either amylase secretion or calcium metabolism, they nevertheless open conductance pathways in the acinar plasma membrane mainly per