A covalently cross-linked complex of yeast cytochrome c and beef heart cytochrome c oxidase has been prepared by the method of Birchmeier et al. (1976) [Birchmeier, W., Kohler, C. E., & Schatz, G. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4334-4338]. This complex is linked through cysteine-107 of yeast cytochrome c to a cysteine in subunit III of cytochrome c oxidase. The covalently bound yeast cytochrome c blocked the i nteraction of horse heart cytochrome c with the high-affinity binding site for substrate on the oxidase without affecting binding to the low-affinity site. These results along with the findings of Bisson et al. (1980) [Bisson, R., Jacobs, B., & Capaldi, R. A. (1980) Biochemistry 19, 4173-4178] indicate that cytochrome c in the high-affinity binding site occupies a cleft between subunits II and III on the cytochrome c oxidase complex. Covalent binding of cytochrome c in the high-affinity binding site inhibited cytochrome c oxidase activity with reduced cytochrome c as substrate. Complete inhibition was obtained with one cytochrome c covalently bound per oxidase monomer. Thus, the low-affinity site for cytochrome c does not function independently in electron transfer. Covalently bound cytochrome c was able to transfer electron from ascorbate and N,N,N',N'-tetrt-methylphenylenediamine to cytochrome c oxidase with an overall oxidase activity of around one-sixth of maximal. Binding of horse heart cytochrome c in the low-affinity site increased this electron-transfer activity to a level close to that of unmodified cytochrome c oxidase. Cytochrome c bound to the low-affinity site must increase the rate of electron transfer through the high-affinity site by some allosteric mechanism. © 1981, American Chemical Society. All rights reserved.