To clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca2+), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn2+. Mn2+ significantly increases the sensitivity of CANP to Ca2+ but CANP is not active with Mn2+ alone. The structural changes induced by Mn2+ were compared with those induced by Ca2+, and the structure of muCANP, which is active at microM Ca2+, was also examined for comparison. Mn2+ and Ca2+ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca2+ was not observed with Mn2+. Six moles of calcium bound to mCANP and the average dissociation constant of Ca2+ was 150 microM. The structure of muCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of muCANP indicated that the structure of muCANP was looser or SH groups were more exposed than in the case of mCANP. A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca2+ can be explained in terms of conformational change and activation of the active-site SH group of CANP.