In this study, K+ concentration was measured in effluent samples from superfused guinea-pig pancreatic pieces in control conditions and during stimulation with ACh, employing the technique of flame photometry. ACh (10(-7)-10(-5) M) evoked a dose-dependent and sustained increase in K+ concentration in the effluent (K+ release). The removal of Ca2+ from the superfusing medium and the addition of 10(-4) M EGTA caused a significant (P < 0.05) reduction in the ACh-evoked K+ efflux. Replacement of extracellular Cl- in the superfusing physiological salt solution with NO3- abolished the ACh-induced K+ efflux. In contrast, when Cl- was replaced with Br-, ACh still evoked marked K+ release. Pretreatment of pancreatic segments with the loop diuretic furosemide (10(-4) M) resulted in an inhibition of K+ efflux elicited by ACh. Stimulation of pancreatic segments with the Na(+)-K(+)-ATPase inhibitor ouabain (10(-3) M) caused a large efflux of K+. In the continuous presence of ouabain, ACh application elicited no further change in the K+ release. The results indicate that ACh-evoked K+ release from guinea-pig pancreatic segments is sensitive to ouabain, Cl-, furosemide and extracellular Ca2+ and that only the basal efflux is augmented by ouabain. The findings provide further evidence that a diuretic-sensitive coupled Na(+)-K(+)-Cl- cotransport system operates in the guinea-pig pancreas, as it does in other similar transporting epithelia, to bring about K+ mobilization.