We describe here a technique using bolus 2-[3H]deoxyglucose (2-[3H]DG) administration for estimating the glucose metabolic rate (R'g) in individual tissues of the rat during exercise. After 50 min of treadmill running at a moderate work load various tissues were analyzed for accumulation of phosphorylated 2-[3H]DG and/or glycogen mass. There was considerable heterogeneity in R'g in response to exercise among different muscles, despite similar basal values. R'g increased 32-fold above basal in soleus (to 90.4 +/- 5.7 mumol X 100 g-1 X min-1), 42-fold in red gastrocnemius (to 71.2 +/- 1.6), 5-fold in white gastrocnemius (to 7.0 +/- 0.8), and 2-fold in extensor digitorum longus (to 4.1 +/- 1.0) during exercise. There was a close relationship between muscle glycogen depletion and R'g within different muscles. In view of the magnitude of the increase in R'g during exercise, this method provides a very sensitive index of muscle fiber recruitment during exercise. The pattern of exercise-stimulated R'g in muscles of different fiber composition was similar to that seen with insulin stimulation. However, moderate exercise alone produced increments in R'g (soleus and red gastrocnemius) that were significantly higher (P less than 0.005) than that observed during maximal insulin stimulation even though whole body glucose utilization was slightly higher during maximal insulin stimulation. These data emphasize the heterogeneity of the response in different muscles during exercise and suggest that intracellular events beyond glucose transport may be rate limiting under the influence of one or both of these stimuli.