This study investigated the effects of exercise training duration on the myosin heavy chain (MHC) isoform distribution in rat locomotor muscles. Female Sprague-Dawley rats (120 days old) were assigned to either a sedentary control group or to one of three endurance exercise training groups. Trained animals ran on a treadmill at similar to 75% maximal Og uptake for 10 wk (4-5 days/wk) at one of three different exercise durations (30, 60, or 90 min/day). Training resulted in increases (P < 0.05) in citrate synthase activity in the soleus and extensor digitorum longus in both the 60 and 90 min/day duration groups and in the plantaris (Pla) in all three exercise groups. All durations of training resulted in a reduction (P < 0.05) in the percentage of MHCIIb and an increase (P < 0.05) in the percentage of MHCIIa in the Pla. The magnitude of change in the percentage of MHCIIb in the Pla increased as a function of the training duration. In the extensor digitorum longus, 90 min of daily exercise promoted a decrease (P < 0.05) in percentage of MHCIIb and increases (P < 0.05) in the percentages of MHCI, MHCIIa, and MHCIId/x. Finally, training durations greater than or equal to 60 min resulted in an increase (P < 0.05) in the percentage of MHCI and a concomitant decrease (P < 0.05) in the percentage of MHCIIa in the soleus. These results demonstrate that increasing the training duration elevates the magnitude of the fast-to-slow shift in MHC phenotype in rat hindlimb muscles.