Reduction in oxygenation induces inhibition of Na+/K+ ATPase in a number of cells and tissues, including hepatocytes. When not reversed, decrease in Na+/K+ pump activity leads to a gradual Na' accumulation, cell swelling and death. However, when accompanied by suppression of dissipative cation pathways, it has also been shown to be a beneficial adaptive strategy used by some hypoxia-tolerant species to reduce ATP consumption during prolonged periods of anoxia. This study aims to investigate acute hypoxic responses of the Na+/K+ ATPase in primary cultures of trout hepatocytes. Gradual decrease in oxygenation was followed by an instantaneous transient dose-dependent downregulation of the Na+/K+ ATPase transport activity, but was without an effect on hydrolytic function of the enzyme. Hypoxia-induced inhibition of active K+ influx was reversed spontaneously when hypoxic incubation time exceeded 20 min. The stimulating effect of prolonged hypoxic exposure on the Na+/K+ pump is most probably secondary to hypoxia-induced activation of the Na+/H+ exchanger with the following Na+ accumulation leading to Na+/K+ ATPase activation. Hypoxia-induced inhibition of the Na+/K+ pump was not caused by ATP depletion or global oxidative stress. However, local controlled production of reactive oxygen species seems to play an important role in hypoxia-induced regulation of the Na+/K+ ATPase. Treatment of cells with mercaptopropionyl glycine (MPG), a scavenger of OH-, abolished hypoxia-induced inhibition of the Na+/K+ ATPase. Earlier on we have shown that activation of Na+/H+ exchanger under hypoxic conditions can be opposed by MPG treatment as well. Taken together our results suggest that regulation of both oxygen-sensitive transporters may be accomplished by local changes in free radical production.