PURPOSE: Mitochondria play a principal role for metabolism and have a primary role in regulating respiration in myocytes. Recently, we have shown that the muscle-specific protein myoglobin (Mb) interacts with complex IV to augment mitochondrial respiratory capacity in skeletal muscles. However, the precise mechanism for the Mb-mediated upregulation remains unclear. The present study has focused on effect of the endurance training (eTR) on Mb within the mitochondria. METHODS: Wistar male rats aged 10-week, were subjected to eTR sessions for 4 weeks (25 m/min X 60 min X 5-day/week, n=6). Muscle specimen from the deep portion of m. Gastrocnemius was taken and homogenized. Crude mitochondria were isolated by differential centrifugations and washed with the mitochondrial isolation buffer. The isolated mitochondria were treated with proteinase K (PK), osmotic shock (OS), and SDS (or TriX) in order to digest proteins on the outer membrane and in the intramembrane. The final samples were subjected to SDS-PAGE and immunoblotting using antibodies to localize the proteins in the mitochondria. RESULTS: The eTR increased VDAC-I and COX-IV around +80~130% as compared with non-exercise control (p<0.05)., Mb increased by +50% (p<0.05). Western blotting analysis revealed that the PK digested Tom20, and Tom20 band intensity decreased with the amount of PK used. PK treatment, however, did not affect Mb found in the mitochondrial fraction. Combining treatment with PK, OS and SDS (or TrX) allowed immunoblotting detection of the mitochondrial proteins localized in specific regions of the mitochondria. Mb was detected with either PK or OS treatment. But it cannot be detected with a combined PK+OS treatment, suggesting that Mb associated with the inner membrane (intramembrane side, not matrix side) of the mitochondria. The Mb content inside the mitochondria in eTR rat was similar with that in the control muscles (n.s). CONCLUSION: The present results suggest that Mb in muscle cells localizes both in the cytosol and in the mitochondrial intermembrane space. Although eTR elevates mitochondrial volume and Mb content but does not change Mb content in the mitochondria. Therefore, the observation might imply that the dynamic flux of Mb from cytosol to mitochondria has greater importance than just the amount found in the mitochondria