Abstract. The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 #m 3, an inner membrane area of 6.5 #m 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of-7,500/#m 2 and occupy ~40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 ,~,) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.T HE molecular structure of mitochondria and in particular of their inner membrane is of great interest to bioenergetics because most of the enzymes involved in energy transduction are associated with these organelles (7). Many of the energy-transducing enzymes, notably the enzymes of the respiratory chain and the H÷-ATPase, are multisubunit complexes which span the membrane and represent ~80% of the total intrinsic protein mass of this membrane (21). Pictures of freeze-fractured inner membranes show a dense packing with intrinsic proteins (19,46). How dense the various protein complexes are built into the inner membrane, and how they are distributed along the plane of the membrane is important for the understanding of the mechanism of energy-transducing reactions in mitochondria.This sort of information is scarce due to the complexity of the mitochondrial ultrastructure and owing to the fact that it is not accessible through biochemical techniques alone. For instance, Gear and Bednarek (15) used a Coulter counter technique to estimate the number and volume of mitochondria in suspension and related these parameters to biochemical data. The Coulter counter method, however, measures only particle size, but cannot give information on the intramitochondrial structures relevant for bioenergetics, such as inner membrane and matrix. In this paper we present an approach which combines biochemical with stereological techniques so as to obtain parameters describing the molecular infrastructure of mitochondria and in particular of the inner membrane. Stereological procedures were applied on electron micrographs of purified preparations of rat liver mitochondria, to estimate mitochondrial number and volume, matrix volume, and surface areas of inner and outer memb...