Three-dimensional image reconstruction has been applied to electron micrographs of noncrystalline, negatively stained ribosomes obtained from Escherichia coli. Several independent reconstructions all show an overall appearance resembling models that had been derived earlier by direct visual interpretation of electron micrographs. The reconstructed ribosomes show numerous structural details not recognized previously, some of which may be functionally significant. A large elongate cavity (approximately 8-nm long x 5-nm wide x 6-nm [maximal] deep) is present on the surface of the ribosome near the base of its stalk and is identifiable as a portion of a feature termed the interface canyon, which was detected in prior reconstructions of the large ribosomal subunit (Radermacher, M., T. Wagenknecht, A. Verschoor, and J. Frank. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1107-1114). On the back of the ribosome, near the base of the central protuberance, is a hole leading to the interface canyon, which likely represents an exit site for the elongating polypeptide produced during protein biosynthesis. The exposed portion of the interface canyon appears well suited to bind two tRNA molecules in a configuration that is consistent with biochemical and structural data on the mechanism of peptide bond biosynthesis.