High-Ca2+-requiring calcium-activated neutral protease (mCANP), a dimeric enzyme composed of large (Mr = 80,000) and small (Mr = 28,000) subunits, is resistant to carboxypeptidase Y (CPase Y) in the absence of NaSCN. In the presence of 0.2 M NaSCN, CPase Y digested mCANP, one or two amino acids being released from the COOH-termini of the large and small subunits, but no change occurred in the activity of the digested mCANP. In the presence of 1 M NaSCN, 8-10 amino acids were released from the subunits by CPase Y, and the COOH-terminal potential Ca2+-binding sites of both subunits were destroyed. On digestion under these conditions, mCANP lost the ability to form a complex, and the proteolytic activity was not recovered even when the digested subunits were mixed with native subunits. These results suggest that the COOH-terminal regions of the two subunits of mCANP, which constitute the helical portions of the COOH-terminal E-F hand structures in both subunits, are essential for the subunit association and resulting proteolytic activity.