The calcium-binding characteristics of the EDTA-extractable proteins (EEP) from calf lens fiber membranes were studied by equilibrium dialysis and far-ultraviolet circular dichroism measurements. The EEP proteins appeared to contain binding sites with different affinities for calcium. These sites seem to behave as positively cooperating Ca2+binding sites with a total capacity of 25 mol Ca2+per mol EEP. The mean apparent dissociation constant (KD) for the Ca2+binding sites was determined to be 7.7μM. Calcium binding probably is accompanied by a decrease in the apparentα-helical content of the EEP proteins.The present results indicate that the EEP proteins belong to the group of proteins possessing high affinity and binding capacity for calcium. Because of the high calcium-binding capacity, the EEP proteins possibly function as an intracellular calcium store in the lens. The calcium-sensitivity of the conformational state of the EEP proteins, however, might point to a possible regulating function of these membrane proteins in calcium-dependent cellular processes in the lens.