To preserve thermoneutrality, cold exposure is followed by changes in energy expenditure and basal metabolic rate (BMR). Because nitric oxide (NO) modulates mitochondrial O₂ uptake and energy levels, we analyzed cold effects (30 days at 4°C) on rat liver and skeletal muscle mitochondrial NO synthases (mtNOS) and their putative impact on BMR. Cold exposure delimited two periods: A (days 1-10), with high systemic O₂ uptake and weight loss, and B (days 10-30), with lower O₂ uptake and fat deposition. mtNOS activity and expression decreased in period A and then increased in period B by 60-100% in liver and skeletal muscle (P < 0.05). Conversely, mitochondrial O₂ uptake remained initially high in the presence of L-arginine and later fell by 30-50% (P < 0.05). On this basis, the estimated fractional contribution of liver plus muscle to total BMR varied from 40% in period A to 25% in period B. The transitional modulation of mtNOS in rat cold acclimation could participate in adaptive responses that favor calorigenesis or conservative energy-saving mechanisms.