The impact of tolerance on the metabolism of the whole body, skeletal muscle, brain, kidneys, splanchnic region, and heart during prolonged pentobarbital anesthesia was evaluated in 80 dogs. Oxygen consumption (&OV0312;o2) for each organ system and whole body was calculated from measured blood flow rate and the difference in blood oxygen content between arterial and venous blood during four periods of continuous and unvarying deep pentobarbital anesthesia: 0–3 h, 3–6 h, 12–15 h, and 21–24 h.&OV0312;o2increased with time in whole body (12%), gastrocnemius muscle (83%), calculated entire skeletal muscle (15%), brain (27%), kidneys (20%), and splanchnic area (10%); it decreased in the heart (20%). In all studies, the electroencephalogram indicated a constant deep burst-suppression level of 2–6 bursts/min and blood pentobarbital levels ranged from 4.5–6 mg/dl. About one-fifth of the increase in gastrocnemius &OV0312;o2could be accounted for by the effect of a continuous infusion of succinylcholine, and about two-thirds of the rise in renal &OV0312;o2by increased renal function. The decrease in heart &OV0312;o2was associated with increased cardiac output and decreased systemic vascular resistance.The sustained increase in metabolism was significant and otherwise unexplained in whole body, skeletal muscle, and the brain; it occurred after 3 h and continued through 24 h of pentobarbital anesthesia. This was presumably due to tolerance, and was manifested as increased metabolism during steady deep anesthesia with unchanged blood levels of pentobarbital rather than as a greater requirement for pentobarbital.