This investigation evaluated the hypothesis that the age-related decline in cold-induced thermogenesis observed in male (F344) rats is associated with altered substrate concentrations of glucose, lactate, and/or liver and muscle glycogen. Body mass-independent O2 consumption, core temperature, and serum glucose and lactate concentrations were measured at rest and during 4 h of exposure to 5 degrees C in male F344 rats ages 6, 12, and 26 months. At the end of the 4-h cold exposure, liver, soleus, and gastrocnemius tissues were removed, frozen, and analyzed for glycogen concentration and/or citrate synthase activity. Core temperature decreased during cold exposure and was consistently less in the 26-month versus the 6- and 12-month rats. There were no significant differences between the 6- and 12-month-old rats with respect to cold-induced O2 consumption, but measures were significantly lower in the 26-month-old rats. During cold exposure, serum lactate and glucose concentrations increased in the 26-month-old animals compared to those in the 6- and 12-month-old rats, while liver glycogen concentrations decreased in all groups, and gastrocnemius glycogen contents decreased in the 12- and 26-month-old rats. Citrate synthase specific activity (mumol.[min.microgram.protein] -1) did not differ with age. These data suggest that carbohydrate availability (as measured by serum glucose and muscle glycogen) is not a limiting factor in the attenuated cold-exposed thermogenic response of the 26-month-old male F344 rat. However, it appears that the 26-month-old rat may have a diminished capacity to fully oxidize carbohydrate during cold exposure.