Cross-sectional area (CSA), succinate dehydrogenase (SDH), and α-glycerophosphate dehydrogenase (GPD) activities were measured in single fibres of adult rat medial gastrocnemius muscle (deep region) using quantitative histochemical procedures. The same fibres were identified in serial sections stained by immunohistochemistry with monoclonal antibodies specific for selected myosin heavy chain (MyHC) isoforms. The values of CSA, SDH and GPD activities formed a continuum, but significant differences in mean values were observed among fibre types of presumed homogeneous MyHC content. Type IIA fibres were the smallest, type IIB fibres were the largest, and type I and IIX fibres were intermediate. Type IIA fibres had the highest SDH activity, followed in rank order by type IIX, type I and type IIB. The average GPD activity was ranked according to fibre type such that IIB>IIX>IIA>I. Hybrid fibres co-expressing two MyHC isoforms generally showed intermediate mean CSA, SDH a nd GPD values lying between their respective pure MyHC fibre types. Across all fibres, there was an inverse relationship between SDH activity and CSA and between GPD and SDH activities, and a positive correlation between GPD and CSA. Moreover, a significant interdependence between CSA, SDH activity, GPD activity and MyHC content existed on a fibre-to-fibre basis, suggesting that the MyHC isoform expressed in a fibre is associated with differences in size, oxidative and glycolytic capabilities of muscle fibres. In fact, most of the fibres could be discriminated into discrete groups with the same MyHC content when their CSA, SDH and GPD values were considered together.