To assess the mechanism of insulin resistance in metabolic acidosis, we investigated insulin receptor binding and glucose uptake in isolated rat epididymal adipocytes at three pHs, acidic (pH 6.8), neutral (pH 7.4) and alkaline (pH 8.0) groups. After male Sprague-Dawley rats weighing 220-240 g were submitted under chloral hydrate anesthesia, epididymal fat pads were dissected and isolated. [125I]-insulin binding rate was not different between alkaline and neutral groups, but that of acidic group was markedly decreased to approximately 30% of neutral group at maximum specific insulin binding rate. Scatchard analysis showed that numbers of insulin receptors were not different among three groups, but values of binding affinity in acidic group was significantly lower than those of the others. 2-[3H]deoxy-D-glucose uptake of alkaline and neutral groups were increased to reach steady state immediately, but that of acidic group increased slowly and reached at maximum which was lower than the other two groups. Furthermore, 2-deoxy-D-glucose uptake against amount of bound insulin was lower in acidic group than other two groups. From these results, it is suggested that insulin resistance in metabolic acidosis resulted from the combined defects in binding affinity and post-binding process in the insulin receptor.