Carbohydrate metabolism in exercise, including regulation of glucose production, was studied by isotope-dilution methods, and these were evaluated. Chronically catheterized rats were examined before, during, and after 45 min of running at either low (LIE) or moderate (MIE) intensity. Glucose production (Ra) and disappearance (Rd), as well as muscular glycogen breakdown (Gly), were estimated by primed constant infusions of [3-3H]- and [U-14 C]glucose, and pyruvate oxidation was estimated by sampling of expired 14CO2. During exercise, Ra increased faster than Rd and was, as were steady-state glucose concentration (G) and Gly, directly related to exercise intensity. During recovery Ra and G decreased rapidly, but after MIE, G showed a rebound increase. 14C estimates and chemical measurements sometimes disagreed. Methodological evaluation showed marked incorporation of label in glycogen, lipid, and protein at rest and mobilization of label during exercise. 14CO2 recovery in expired air ranged from only 50% at rest to 77% during MIE. In conclusion, during exercise, mobilization of hepatic glycogen is a primary event and not secondary to increased muscular demand. During and after exercise, plasma glycogen is not precisely controlled at euglycemic levels. Isotope methods may be used to study carbohydrate metabolism in exercising rats, but the results (especially 14C data) should be interpreted with caution.