Rat thymocyte glucose transport activity was measured by the uptake of 3H-2-deoxyglucose. In the presence of 5mM glucose, DNP stimulated the uptake of 2-deoxyglucose. In the absence of glucose, DNP inhibited the uptake of 2-deoxyglucose. Both stimulatory and inhibitor effects of DNP were DNP concentration dependent. The incubation of thymocytes with nutrients which are metabolized through mitochondrial oxidation, such as beta-hydroxybutyrate, pyruvate, alanine and glutamine, did not support the stimulatory effects of DNP on 2-deoxyglucose uptake. The incubation of thymocytes with a nutrient which can support glycolysis, such as adenosine, supported the stimulatory effect of DNP on 2-deoxyglucose uptake. Iodoacetate, an inhibitor of glycolysis, inhibited 2-deoxyglucose uptake even in the presence of glucose. The present data suggested that the stimulatory effect of DNP on thymocyte 2-deoxyglucose uptake required intact glycolysis activity.