Three of 46 patients with autoimmune hemolytic anemia (AIHA) satisfied the diagnostic criteria for mixed-type AIHA in which both warm-type and cold-type autoantibodies against red blood cells (RBCs) are present. The specificities of these autoantibodies were analyzed. All of the warm-type autoantibodies were IgG-chi, and the specificities were not serologically classified. The autoantibody of patient 1 reacted to the 41- and 80-kD peptides on immunoblotting, and the epitope corresponding to it was papain sensitive. Two warm-type autoantibodies from patients 2 and 3 resembled each other in serological analyses and reacted with a protease- and neuraminidase-resistant antigen. However, the antigen corresponding to the autoantibody of patient 3 was located on the 37-kD peptide by immunoprecipitation. All of the cold-type autoantibodies were IgM-cha and showed high titer and high thermal amplitude. According to the reaction pattern with untreated and enzyme-treated RBCs, the cold-type autoantibodies of patients 2 and 3 were revealed to be anti-Om and anti-I, respectively. In patient 1, the cold-type autoantibody was characterized as having high affinity for autologous RBCs, but its specificity was unclassified. The antigens corresponding to the cold-type autoantibodies were not located by immunoblotting and immunoprecipitation. These serological and immunochemical approaches to autoantibodies in mixed-type AIHA revealed that the warm and cold components recognized the different antigens.