The stapedius muscle (SM) is supposed to prevent cochlear damage by noise. Consequently functional demands are the ability of fast contraction with long endurance. This implies the presence of a large fraction of myosin type II fibres with an appreciable oxidative capacity. We determined the myosin composition of SM fibres using consecutive complete SM cross-sections (6 week old rats) which were processed by enzyme histochemistry (EHC) to determine acid/alkali lability of myofibrillar adenosine triphosphatase (mATPase) or by immunohistochemistry (IHC) using myosin heavy chain (MyHC) antibodies. Method accuracy was determined in co-processed extensor digitorum longus (EDL). Four hundred SM and 200 EDL fibres were assigned to mATPase type I, IIA, IIB, IIX or 'miscellaneous' ('Misc') categories. Per mATPase category the fibres were attributed to groups with specific MyHC composition. In the EDL, mATPase type I and IIB fibres expressed only MyHC I and IIB respectively, whereas about 10% of the type IIA and 40% of the type IIX fibres expressed more than one MyHC. Thus IHC detects amounts of myosin isoforms which are not detected by EHC. The mATPase IIX category criterion leaves the possibility that this category contains fibres with myosin type IIA and/or IIB in larger amounts. The criteria of the mATPase categories type I, IIA or IIB preclude assignment to these categories of fibres which also contain other myosin isoforms in larger amounts. Such fibres were classified in one of the mATPase 'Misc' categories. Thus in the EDL the capability of the EHC criteria to select 'pure' fibres in terms of myosin differs per mATPase category. None of the SM fibres were assigned to the mATPase type I or IIB categories, about 25% to the type IIA, 60% to type IIX and 15% (including most fibres which expressed MyHC I) to a 'Misc' category. All SM fibres expressed two or more MyHC isoforms, MyHC IIB occurring in all fibres and substantial amounts of MyHC IIA and/or IIX in most. These findings confirm the hypothesis that such fibres have the capacity to contract fast and have the better fatigue resistance.