Simultaneous determination of proton uptake and oxygen binding has been carried out on Helix pomatia beta-hemocyanin under equilibrium conditions in the absence of buffer and at different initial pH values. Oxygen-binding isotherms of unbuffered H. pomatia beta-hemocyanin, in the presence of phenol red as pH indicator, have been determined employing a thin-layer apparatus. Application of this very accurate technique allows monitoring of proton uptake (or release) coupled to O2-binding also at extremes of saturation which are often difficult to explore and analyze. The data have been analyzed within the framework of the cooperon model (M. Brunori, M. Coletta and E. Di Cera, Biophys. Chem. 23 (1986) 215) and compared with those obtained in the presence of buffer. Comparison of pH changes with ligand binding of the T state over all the saturation range has allowed us to discriminate and obtain quantitative estimates of the Bohr protons associated with both oxygenation of the T state and quaternary allosteric transition; no protons are taken up or released during oxygenation of the R state. These results differ quantitatively from those obtained in the presence of buffer, which alters significantly the T state contribution to the overall Bohr effect.