We have examined the competitive binding of oxygen and carbon monoxide to the multisubunit hemocyanin of the tarantula Eurypelma californicum. Employment of high-precision thin-layer methods has enabled detailed characterization of the pure oxygen and pure carbon monoxide binding curves, as well as binding curves performed under mixed-gas conditions. The pure oxygen binding curve and the displacement of oxygen by carbon monoxide at full ligand saturation are highly cooperative, but in the absence of oxygen, carbon monoxide binds noncooperatively. The results were analyzed globally within the framework of a nested allosteric model [Robert, C.H., Decker, H., Richey, B., Gill, S.J., & Wyman, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1891-1895] which takes into account the hierarchy of subunit structure present in the macromolecule. The use of two ligands enables one to recognize two distinct levels of allosteric interaction functioning in the protein assembly. The binding characteristics of the allosteric states demonstrated for Eurypelma follow a similar pattern as those found earlier for Homarus americanus.