Introduction. Our recent study on the direct comparison of the antigenicity of free human hemoglobin (Hb A) and Hb A bound by rabbit haptoglobin (Hp) revealed that antigenicity of Hb A is somewhat increased by association with Hp.l) It has been generally observed that Hb A has an abnormally weak antigenicity. Only when large amount of Hb A was injected into rabbits, sufficient antibody was obtained.2> From the structural property having a molecular weight of 68,000, being rich in a-helix, having hemes as prosthetic group, and having a sufficient polarity, however, the weak antigenicity of Hb A is difficult to be explained. Ryan and Lee, on the other hand, reported that weak antigenicity of Hb A depends on its easy digestibility by lysosomal enzyme, cathepsin.3~ In this connection, the present investigation was performed to clarify one of the mechanisms of increased antigenicity of Hb A bound by Hp by comparing the susceptibility of free Hb A and Hb A-Hp complex to various proteases. Materials and methods. Papain and pepsin, twice recrystallized, were purchased from Sigma, trypsin from N.B.C., and casein nach Hammersten from Merck. Cathepsin was prepared from the bovine spleen according to the method of Ragab et al.4> Fluorescein isothiocyanate, 10% on Celite, was obtained from Calbiochem, Los Angeles, Calif. Preparation o f Hb-Hp complex. Hp and Hb-Hp complex were prepared by the method of Hamaguchi.5~ Preparation o f Hb *-Hp complex. Fluorescein-hemoglobin (Hb*) was prepared by the method of De Lumen et al.° and passed through the Sephadex G-100 column. After the addition of purified Hp, fluorescein-hemoglobin-haptoglobin complex (Hb *-Hp complex) was separated from free Hb* by passing through the Sephadex G-100 *' This investigation was aided by Grant No. 7058 (1971), No. 87036 and No. 757037 (1972) from the Ministry of Education of Japan .