AbstractA technique is described for the isolation and purification of intact, respiratory‐competent mitochondria from Sachizo‐saccharomyces pombe. The purified mitochondria are capabel of oxidizing NADH and succinate as resporatory substrates. indicating the presence of succinate dehydorgenase and an NADH dehydrogenase located on the outer suface of the inner membrane. Mitochondria display good respiratory control with an ADP/O ratio of <2. Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched repiratory pathway to molecular oxygen. Immunogold labelling using antisera raised against mitochodria proteins (SSPI, SSCI, and PHSPI) from three different species, namely S. pombe, Saccharomyces cerevisiane and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure. The immunocytochemistry was carried out using cells containing wild‐type levels of SSPI protein and cells over‐expressing the protein. These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo. In vitro import experiments using COXIV‐DHFR indicate that purified S. pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential.