Washed rainbow trout erythrocytes incubated at 14 degrees C in Eagle's minimal essential medium and Cortland saline displayed sharp reductions in volume and water content, nucleoside triphosphate, K+ and Cl- concentrations. Mg2+ and, to a lesser extent, Na+ concentrations increased. Cellular to medium Cl- ratios were indicative of membrane hyperpolarization. Morphological irregularities were also observed. Oxygen consumption and hemoglobin system organization were not grossly affected. Supplementation with pyruvate stabilized nucleoside triphosphate concentrations for at least 24 hr, and reduced rates of volume and compositional change to some extent. Addition of norepinephrine at physiologically realistic levels led to stabilization of Cl- content and reductions in Mg2+ accumulation and water loss. Transient but modest increases in K+ and Ca2+ were coupled, under these circumstances, with some decrease in Na+ concentration. Factors which may contribute to the dysfunctional status of these cells in vitro are discussed.