To assess the mechanism of the accelerated gluconeogenesis in the insulin-deficient state, chronically catheterized (carotid artery, portal vein, hepatic vein, vena cava) normal (C; n = 9) and depancreatized (PX; n = 7) dogs were studied during rest (40 min) and moderate exercise (150 min). Tracers ([14C]alanine, [3H]glucose) and dye were infused to measure determinants of gluconeogenesis in the gut and liver. Arterial levels, net gut output, hepatic load, and net hepatic uptake of alanine were similar in C and PX at rest. During exercise, alanine levels fell in C but rose approximately 100% in PX. Exercise did not affect gut output or liver uptake of alanine in C but increased these variables by approximately 50 and 100% in PX due to an increase in hepatic alanine load. Arterial lactate was similar at rest in C and PX but rose fourfold more in PX with exercise. Net gut lactate output was fivefold greater in PX during rest and exercise. Net hepatic lactate uptake was present in PX at rest, whereas net output was evident in C. In response to exercise, hepatic lactate uptake was increased further in PX due to a rise in hepatic lactate load. Net hepatic lactate uptake was not evident until the end of exercise in C. Net hepatic glycerol uptake was elevated at rest in PX and during the initial 60 min of exercise due to an elevated hepatic load. In contrast to the high rates of gut lactate and alanine output in PX, gut glycerol output was not present. Gluconeogenesis from lactate and alanine was 5- to 10-fold higher in PX than C during rest and exercise. At rest, this resulted, in part, from a twofold greater intrahepatic gluconeogenic efficiency. During exercise, the greater conversion occurred even though efficiency was not consistently greater. In summary, gluconeogenesis from alanine, lactate, and glycerol in the insulin-deficient diabetic state 1) is exaggerated at rest, due to an increased capacity for hepatic lactate extraction, increased hepatic precursor loads, and a greater gluconeogenic efficiency; 2) is accelerated further by exercise due to added increments in hepatic precursor loads; and 3) is exaggerated partly because of a greater net gut alanine and lactate output.