Variously modified protein antigens were tested by footpad assay to clarify the effect of these medications in producing delayed hypersensitivity in mice. The most potent antigen examined was carboxyl-methylated serum albumins. These antigens were highly basic proteins and hydrophobic compared with native serum proteins. They stimulate humoral antibody response in mice poorly, and remain at the subcutaneous injection site much longer than native serum albumins. In vitro tests of susceptibility of thymus and spleen cells and peritoneal macrophages to the antigens revealed that methylated serum albumins possessed the stimulatory activity to the latter and were toxic to the former. As for macrophage, fluorescein-labelled methylated serum albumin showed an affinity to their membrane and were phagocytosed, but FITC-BSA did not show any affinity to the macrophages. These biological activities to tissue or cells may be contributable to render methylated serum albumins to induce and elicit delayed hypersensitivity preferentially in mice.