The purpose of this investigation was to examine critically the validity of a computerized quantitative microphotometric histochemical technique for the determination of succinate dehydrogenase (SDH) activity in skeletal muscle fibres. Sections from the anterior costal diaphragm were removed from Fischer-344 rats (n = 12) and assayed histochemically to determine SDH activity. The SDH activity in individual muscle fibres was computed using a computerized microphotometric histochemical technique which involves measurement of the optical density of deposited diformazan derived from nitroblue tetrazolium within the fibres. To validate the histochemical technique, whole muscle SDH activities were calculated from the histochemical procedure and were compared to SDH activities determined from whole muscle homogenates via a standard quantitative biochemical assay. The mean within-day variability of the computerized microphotometric histochemical technique of determining SDH activity was 6% (range = 0.5-10.9%) for an area containing ~50 fibres and 6.1% (range = 1.05-14.9%) for an individual muscle fibre. Similarly, the mean between-day variability of the microphotometric histochemical technique of determining SDH activity was 5.9% (range = 2.6-13.9%) for an area containing ~ 50 fibres and 6.6% (range = 2.2-13.9%) for an individual muscle fibre. The inter-class correlation coefficient between biochemically determined SDH activity and histochemically determined SDH activity was r = 0.83 (p (0.05). Collectively, these data demonstrate that the quantitative histochemical technique of Blanco eta]. (1988) is both valid and reliable in the determination of SDH activity in skeletal muscle fibres.