The binding constants, K(1) and K(2), and the number of Ca(2+) ions in each of the two high affinity sites of Ca(2+)-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K(1) and K(2), it was found that two hydrogen ions are released for each Ca(2+) bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K(s), for the binding of Ca(2+) to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K(s) and its pH dependence with those of K(1) and K(2) showed that the site corresponding to K(s) is to be identified with that of K(2). This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca(2+) that causes the blue to purple color change.Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.