Manipulations of pH and electrical gradients in a perfused preparation were used to analyze the factors controlling ammonia distribution and flux in trout white muscle after exercise. Trout were exercised to exhaustion, and then an isolated-perfused white muscle preparation with discrete arterial inflow and venous outflow was made from the posterior portion of the tail. The tail-trunks were perfused with low (7.4)-, medium (7.9)-, and high (8.4)-pH saline, achieved by varying HCO3- concentration ([HCO3-]) at constant Pco2. Intracellular and extracellular pH, ammonia, CO2, K+, Na+, and Cl- were measured. Muscle intracellular pH was not affected by changes in extracellular pH. Increasing extracellular pH caused a decrease in the transmembrane NH3 partial pressure (PNH3) gradient and a decrease in ammonia efflux. When extracellular K+ concentration was increased from 3.5 to 15 mM in the medium-pH group, a depolarization of the muscle cell membrane potential from -92 to -60 mV and a 0.1-unit depression in intracellular pH occurred. Ammonia efflux increased despite a marked reduction in the PNH3 gradient. Amiloride (10(-4) M) had no effect, indicating that Na+/H(+)-NH4+ exchange does not participate in ammonia transport in this system. A comparison of observed intracellular-to-extracellular ammonia distribution ratios with those modeled according to either pH or Nernst potential distributions supports a model in which ammonia distribution across white muscle cell membranes is affected by both pH and electrical gradients, indicating that the membranes are permeable to both NH3 and NH4+. Membrane potential, acting to retain high levels of NH4+ in the intracellular compartment, appears to have the dominant influence during the postexercise period. However, at rest, the pH gradient may be more important, resulting in much lower intracellular ammonia levels and distribution ratios. We speculate that the muscle cell membrane NH3-to-NH4+ permeability ratio in trout may change between the rest and postexercise condition.