The effect of glucose, galactose, and valine on intestinal Cl- transport and intracellular Cl-, Na+, and K+ activity was investigated in isolated segments of Amphiuma small intestine. By use of double-barreled Cl- -specific microelectrodes, it was observed that galactose and valine reduced the luminal membrane potential (psi m) and eliminated the difference between the Cl- equilibrium potential (ECl) and psi m, i.e., the Cl- accumulation potential (ECl-psi m) approached zero. Simultaneously, Cl- absorption (JnetCl) was reduced in short-circuited tissues and Na+ absorption was enhanced. In contrast, after exposure to glucose, psi m and ECl-psi m declined only transiently and JnetCl was unaltered. In tissues pretreated with galactose to reduce Cl- transport, addition of glucose to the serosal medium restored Cl- accumulation across the luminal membrane and the Cl- absorptive current. Glucose, galactose, and valine each reduced intracellular K+ activity significantly. Galactose and valine each increased [corrected] intracellular Na activity (aiNa) markedly, whereas glucose increased aiNa only slightly. In conclusion, intestinal ion transport can be limited by the availability of metabolic substrate. The nonmetabolized solutes galactose and valine inhibited Cl- uptake and net Cl- absorption while stimulating net Na absorption, as though net Na+ absorption has priority over Cl- transport at the cellular level. Cl- transport is reduced at both mucosal and serosal membranes. At the luminal membrane electrogenic Cl- uptake is slowed or a backleak of Cl- is enhanced; at the serosal membrane Cl- exchange with Na+ (and HCO3-) driven by the Na+ gradient is reduced. The availability of metabolizable glucose to the cell prevents the reduction in net Cl- absorption.