Subunit III, the 8 kDa component of the chloroplast CFo H+ channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca(2+)-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrene-divinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, the psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca(2+)-ligand blot assay known to detect high affinity Ca(2+)-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl2 in the presence of 10-fold excess of MgCl2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La3+. Ca2+ binding was maximum at pH 7.5-8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca(2+)-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca(2+)-binding site. Based on the accepted models for the hairpin conformation of the subunit III, it does seem clear that the Ca(2+)-binding site can form on the lumenal side of the membrane in the functional CFo structure.