Binding of carbon monoxide to the separated α and β chains of hemoglobin, with and without bound p-mercuribenzoate, has been measured at temperatures from 5 to 340 K for times 2 µs to 1 ks using flash photolysis. All four proteins exhibit three different rebinding processes. The data are interpreted by a model in which the carbon monoxide, moving from the solvent to the binding site at the ferrous heme iron, encounters three barriers. The temperature dependences of the three processes yield activation enthalpies and entropies for the three barriers for all four proteins. Binding at temperatures below about 200 K is nonexponential, implying that the innermost barrier has a distribution of activation enthalpies. The distributions for the four proteins have been determined. At temperatures below 30 K, the CO binding rates approach finite low-temperature limits; binding thus proceeds by quantum-mechanical tunneling. Invoking a simple model, the widths of the innermost barriers are extracted from the measured tunneling rates. The experimental parameters are correlated with structural features of the hemoglobin chains and compared with previously published data on myoglobin and protoheme. A correlation is established between the height of the innermost barrier and the equilibrium CO pressure. © 1978, American Chemical Society. All rights reserved.