Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four microCi of glutamine-U-14C were added to media containing 5 mM or 65 microM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 microM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.