The quantitative assessment of enzyme activities in situ in single muscle fibres is essential for understanding the functions of skeketal muscles. The function of the lateral pterygoid muscle (LPM) is not fully understood because it is a deeply located masticatory muscle and cannot be dissected in an intact configuration. Here we report how to measure the activities of mitochondrial succinate dehydrogenase (SDH) in single muscle fibres in situ in the LPM in sections of rat heads. Unfixed head sections were incubated on gel films containing SDH substrate and nitroblue tetrazolium. During incubation, images of the section due to deposition of the final reaction products, formazans, were captured at intervals of 10 s using a real-time image analysis system for absorbance measurements. We found that the belly of the LPM consisted of four areas with different mean activities of SDH. The lateral and upper areas of the muscle showed similarly high SDH activities. Mean activity in the lower area was the lowest, about half of those of the lateral and upper areas. These results agree with the hypothesis that the superior head of the LPM participates in more continuous contraction and is more resistant to fatigue than the inferior head.