A 193-fold purification of Ca(2+)-binding protein from rat liver mitochondrial matrix was achieved. The Ca(2+)-binding protein consisted of 3 polypeptide subunits whose respective molecular weights by SDS polyacrylamide gel electrophoresis were 62K, 49K and 37K. The molecular weight of the protein was 150K to 220K. The Kd for Ca(2+) was 1.3×10-(5)M and lower for Mn(2+) and Mg(2+). The protein was inactivated by heat treatment at 100℃ for 1 min, though stable against treatment with 0.5% w/v trypsin at 37℃ for 30 min. Ruthenium red did not inhibit Ca(2+)-binding.