A nuclear human blood platelets have been used to study mitochondrial topoisomerase activity in the absence of nuclear contamination. Previous work utilizing this novel system demonstrated that platelet mitochondria contain type-I topoisomerase (Kosovsky, M.J. and Soslau, G. (1991) Biochim. Biophys. Acta 1078, 56-62). The present work has demonstrated that mitochondrial topoisomerase activity was inhibited by the specific topoisomerase-I inhibitor, topotecan, yet was not affected by a specific topoisomerase-II inhibitor, VM-26. These results confirm that platelet mitochondria contain topoisomerase I, yet do not contain a detectable level of topoisomerase-II activity. It has been demonstrated for the first time that antibodies directed against nuclear topo I cross-react with mitochondria topo I. Furthermore, immunoblot analysis of platelet mitochondrial proteins, in conjunction with renaturation studies, has led to the identification of a catalytically active 60-kDa form of mitochondrial topoisomerase I.