A cDNA clone that encodes a novel Ca2+-binding protein was isolated from a human brain cDNA library. The gene for this clone, termed calbrain, encodes a 70-amino acid polypeptide with a predicted molecular mass of 8.06 kDa. The analysis of deduced amino acid sequence revealed that calbrain contains two putative EF-hand motifs that show significantly high homology to those of the calmodulin (CaM) family rather than two EF-hand protein families. By Northern hybridization analysis, an approximate 1.5-kilobase pair transcript of calbrain was detected exclusively in the brain, and in situ hybridization study revealed its abundant expression in the hippocampus, habenular area in the epithalamus, and in the cerebellum. A recombinant calbrain protein showed a Ca2+ binding capacity, suggesting the functional potency as a regulator of Ca2+-mediated cellular processes. Ca2+/calmodulin-dependent kinase II, the most abundant protein kinase in the hippocampus and strongly implicated in the basic neuronal functions, was used to evaluate the physiological roles of calbrain. Studies in vitro revealed that calbrain competitively inhibited CaM binding to Ca2+/calmodulin-dependent kinase II (Ki = 129 nM) and reduced its kinase activity and autophosphorylation.