This study examined the effect of Walker 256 tumor growth in vivo on the metabolism of glucose, glutamine and pyruvate in lymphocytes. A comparison between the metabolism of Walker 256 tumor cells obtained in vivo with that of lymphocytes was also carried out. Lymphocytes and tumor cells were isolated and incubated for 1 h for the following measurements: lactate production from glucose (5.6 mM) and pyruvate (3 mM), glutamate and aspartate formation from glutamine (3 mM) and decarboxylation of [U-14C]-glucose, [U-14C]-glutamine, [1-14C]-pyruvate and [3-14C]-pyruvate. The presence of the tumor increased lactate production (2.7-fold from glucose and 2-fold from pyruvate), decarboxylation of [U-14C]-glucose (3.7-fold) and [1-14C]-pyruvate (4.4-fold) and the formation of aspartate (6.3-fold) and glutamate (4.6-fold) from glutamine. The conversion of glucose to lactate and CO2 was higher in tumor cells as compared to lymphocytes. Tumor cells also showed a higher production of glutamate and an 8-fold increased decarboxylation rate of [U-14C]-glutamine in tumor cells, which was more active than that of lymphocytes even from tumor-bearing rats. Tumor growth stimulated glucose and glutamine metabolism in lymphocytes; however, the importance of this fact for the function of these cells remains to be elucidated.