Recently, significant incorporation of labeled carbon into plasma glucose was documented during infusion of 14C-labeled glutamine in postabsorptive humans. Such labeling of plasma glucose can occur as a result of two different processes: either 1) through incorporation of glutamine carbon into glucose via glutamine entering Krebs cycle at alpha-ketoglutarate or 2) through simple fixation of labeled CO2 resulting from oxidation of labeled glutamine. Therefore, these studies were designed to determine 1) whether glutamine contributes carbon to gluconeogenesis other than through mere CO2 fixation, and, if so, 2) whether the apparent transfer of carbon from glutamine to glucose increases with fasting. Eight healthy adults were studied on two consecutive days: once after an overnight (18-h) fast and again on the second day of fasting (42-h fast). On each study day, subjects received a simultaneous 5-h infusion of D-[6,6-2H2lglucose, L-[3,4-13C2lglutamine, and L-[1-14C]leucine. Apparent rates of incorporation of glutamine carbon into glucose were estimated from the appearance of 13C into plasma glucose; glucose and glutamine production rates (appearance rate [Ra]) were determined from plasma [2H2]glucose and [13C2]glutamine enrichments, respectively. The appearance of 14C into plasma glucose was used to correct the measured rates of carbon transfer from glutamine to glucose as a result of CO2 fixation. We observed that of the apparent contribution of labeled glutamine to gluconeogenesis, only 4% occurred as a result of fixation of labeled CO2, while 96% seemed to occur through other routes. We also observed that between 18 and 42 h of fasting, 1) the relative contribution of protein breakdown to glutamine production was enhanced, while that of de novo synthesis declined; 2) the apparent contribution of glutamine to glucose production rose from 8 +/- 1 to 16 +/- 3% of overall glucose Ra; and 3) the relative apparent contribution of glutamine to gluconeogenesis remained constant. From the current data, it cannot be ascertained to what extent the apparent carbon transfer from glutamine to glucose represents a true contribution of glutamine to gluconeogenesis or mere carbon exchange between the trichloroacetic acid cycle and the gluconeogenic pathway. These findings are nevertheless compatible with a role of glutamine as a significant precursor of glucose in fasting humans.