Microphotometric assay media for the measurement of succinate dehydrogenase (SDH) and cytochrome oxidase activities in sections of human skeletal muscle have been developed. The optimal constitution of these media was determined experimentally. Factors investigated include the effects of substrate concentration, pH, use of different electron acceptors and electron donors, influence of intermediate electron carriers and tissue-stabilizing agents, effects of inhibitors, the extent of endogenous and non-specific reactions and the linearity of the reactions during the time course of the assays. Optimal assay media (SDH) contained 130 mM succinate, 1.5 mM Nitro Blue tetrazolium, 0.2 mM phenazine methosulphate and 1.0 mM sodium azide in 0.1 m phosphate buffer, pH7.0. Cytochrome oxidase was optimally assayed in media containing 4 mM diaminobenzidine and 100 microns cytochrome c. Reactions in individual muscle fibers were found to be linear for incubation times up to 10 min in SDH assays and for more than 15 min in cytochrome oxidase determinations. Some potential uses of these microphotometric assays in the investigation of human metabolic muscle disorders are discussed.