Cytochrome c oxidase (CcO) plays a key role in cellular respiration and energetic metabolism. The latter is a prerequisite for osmotic and synthetic function, motility and the maintenance of cell structure. The objective of this study was to develop and assess a facile microscopic technique that would demonstrate CcO activity insitu, both at the microscopic and sub-microscopic levels. The cytochemical technique proposed is based on oxidation of 3,3'-diaminobenzidine (DAB) by the cytochrome c complex (including CcO), in a chain reaction in which the reagent is polymerized and deposited at the reaction sites. The deposit can be identified by its colour under the light microscope and by osmiophilia under the electron microscope. The reaction is restricted to mitochondria at the light microscope level, and to the outer face of the inner mitochondrial membrane at the ultrastructural level. The activity is inhibited promptly by greater than or equal to 0.5 mM KCN, a specific inhibitor of CcO, and by sodium azide or heat (70 degrees C/5 min). The technique was validated on a number of domestic and laboratory animals, using sperm that were ejaculated or epididymal in origin, cryopreserved or treated. The data obtained displayed activity profiles of individual cells, ejaculates or donors and emphasized differences among species. This technique depicts spontaneous CcO decline during ageing and changes induced by various physical or chemical treatments.