The localization of carbon monoxide oxidase (CO oxidase), the key enzyme in CO metabolism of Pseudomonas carboxydovorans, was examined using modified immunoferritin and protein A-gold techniques. Cell extracts were incubated with specific immunoglobulin G antibodies raised against CO oxidase, followed by treatment with ferritin-conjugated goat-anti-rabbit immunoglobulin G antibodies (pre-embedding labeling). Electron microscopic examination of ultrathin sections showed cytoplasmic membranes and inside-out vesicles labeled at the inner aspect, whereas the outer sides of protoplasts and membrane vesicles remained completely unlabeled. The highly sensitive protein A-gold method has been modified to allow labeling of CO oxidase with good ultrastructural preservation of the bacterial cell. Glutaraldehyde-fixed cells of P. carboxydovorans were osmificated and embedded in glycol methacrylate. Etched ultrathin sections were treated with sodium metaperiodate and incubated with the specific antibodies against CO oxidase. These antibodies were then allowed to react with protein A-gold complexes (postembedding labeling). Exponentially grown cells showed 87% of CO oxidase associated with the cytoplasmic membrane and 13% of the enzyme in the cytoplasm. The results indicate that CO oxidase is attached in vivo to the inner aspect of the cytoplasmic membrane and suggest interaction of the enzyme with a membrane-bound electron acceptor. The ratio of enzyme associated with the cytoplasmic membrane decreased to 50% in the stationary growth phase.