The effect of prolonged (3-5 wk) fasting on tracer-determined glucose turnover and of recycling radioactive glucose has been examined. We followed the specific activity of plasma glucose after the simultaneous administration of 1-14C-glucose and 3-3H-glucose. The rate of glucose turnover decreased during prolonged fasting. Recycling of radioactive glucose was estimated by two different techniques: (1) the appearance of 14C in positions 2 to 6 glucose was measured; (2) the difference in the slopes of specific activity decline for 1-14C-glucose and for 3-3H-glucose was calculated. The two methods of estimating the radioactive recycling gave results similar to each other. The amount of glucose recycled did not change during prolonged fasting. However, in view of the decline in glucose production during fasting, the proportion of glucose production which was represented by recycling increased. Based on weight and urinary nitrogen loss an estimate of the glucose production from amino acids and glycerol was obtained. The difference between the rate of glucose production from the contribution of amino acids and glycerol and that estimated by radioisotopic techniques was much larger than the measured rate of recycling. This finding suggests that either a large exchange of 12C with 14C occurred in some glycolytic intermediates or that a hitherto unknown source of carbon for glucose production appeared during prolonged fasting.