Papain was covalently immobilized onto the surface of porous chitosan beads without or with spacers of different lengths. The relative activity of the immobilized papain was found to be high toward a small ester substrate, N-benzyl-L-arginine ethyl ester (BAEE), but rather low toward casein, a high molecular weight substrate. Papain immobilized with spacer gave an almost constant activity, in marked contrast with the immobilized papain without spacer, whose activity monotonously decreased with decreasing surface concentration. The relative activity of the immobilized papain for hydrolysis of a high molecular weight substrate greatly depended on the length of the spacer. The pH, thermal and storage stabilities of the immobilized papain were higher than those of the free one, and ths papain immobilized directly to the chitosan beads' surface without any spacer gave a higher stability than those immobilized with spacer. The spacer's effect on the activity could be explained in terms of mobility of the immobilized papain molecule.