The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in similar to 500-nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1 degrees or 2 degrees ranging from -60 degrees to +60 degrees. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each similar to 7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of similar to 22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, similar to 14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes similar to 14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion. (C) 1997 Academic Press.