Cubic membranes occur in a variety of membrane-bound organelles in many cell types. By transmission electron microscopy (TEM) these membrane systems appear to consist of highly curved periodic surfaces that fit mathematical models analogous to those used to describe lipidic cubic phases. For the first time, a naturally occurring cubic membrane system has been reconstructed in three dimensions by electron microscopic tomography, and its periodicity directly characterized. Double-tilt tomographic reconstruction of mitochondria in the amoeba, Chaos carolinensis, confirms that their cristae (inner membrane infoldings) have the cubic structure suggested by modeling: studies based on thin-section TEM images. Analysis of the membrane surfaces in the reconstruction reveals the connectivity of the internal compartments within the mitochondria. In the cubic regions, the matrix is highly condensed and confined to a continuous, small space between adjacent cristal membranes. The cristae form large, undulating cisternae that communicate with the peripheral (inner membrane) compartment through narrow tubular segments as seen in other types of mitochondria. The cubic periodicity of these mitochondrial membranes provides an ideal specimen for measuring geometrical distortions in biological electron tomography. It may also prove to be a useful model system for studies of the correlation of cristae-matrix organization with mitochondrial activity.