In our continuing effort to characterize the metal cation binding in bacteriorhodopsin (bR) using Ca(2+)-specific electrodes, potentiometric titration was carried out on deionized solubilized bR (containing monomeric units) and deionized bacterioopsin (bR with its retinal removed). Scatchard plots were analyzed. The monomer was found to have plots similar to those of the trimer, suggesting that the binding sites in bR are localized within the protein monomer unit and not between the molecules within the trimer structure. This also supports the previous assumption that the curvature in the Scatchard plot of regenerated bR is not due to cooperativity of metal cation within the trimer, but rather due to multiple sites. Recent studies further support the finding that the curved Scatchard plot is not due to the cooperativity between the metal ions in the two high affinity sites, wherever they are. The results of the analysis of the Scatchard plot for deionized bacterioopsin have shown a change in the binding characteristics of the high affinity but not the low affinity sites from that observed in bR. This result supports previous conclusions that metal cations in the high affinity sites are not far from the retinal cavity.