The cDNA fragments corresponding to the domains with four consecutive E-F hand structures in the large and small subunits of chicken and rabbit calcium-activated neutral protease (CANP) were inserted into an expression vector (pUC8 or pUC18). The resulting plasmids were used to transform E. coli, and isopropyl-1-thio-beta-D-galactoside (IPTG)-inducible expression was performed. The resulting four kinds of E-F hand structure-domains (the chicken large subunit, rabbit high- and low-calcium-requiring large subunits, and rabbit small subunit) were purified and analyzed for their calcium-binding abilities and capacities by the microscale filter assay. Most of the E-F hand structures could bind calcium and 2 or 4 mol of Ca2+ ions bound to the four consecutive E-F hand structures. The calcium-binding affinity of the E-F hand structures in the large subunit roughly corresponds to the calcium concentration required for its CANP activity.