Three allotypes of mouse factor H, H.1, H.2, and H.3 were purified from the sera of mice with different factor H allotypes, and their functional properties were investigated. The three allotypes all bound to heparin, DNA, Con A, and methylamine-treated mouse C3 (C3(MA)mo) with similar affinities for each protein immobilized, showed identical mobilities on SDS-PAGE, and were reacted well with rabbit polyclonal antibody against H.1 and H.2. Factor I-cofactor activity of these factor H allotypes was measured using highly purified material of mouse, guinea-pig, and human origin. In a homologous system, these allotypes expressed indistinguishable mouse factor I (Imo)-cofactor activity for the cleavage of C3(MA)mo. Imo-cofactor activity was again indistinguishable in these allotypes when methylamine-treated human C3 (C3(MA)hu) or methylamine-treated guinea-pig C3 (C3(MA)gp) was substituted for the C3(MA)mo substrate. The cofactor activity of these factor H allotypes, however, was augmented 4-5 times if C3(MA)hu) was used instead of C3(MA)mo, and was barely detected if C3(MA)gp was employed. In contrast, differences in the potency of the cofactor activity for the three allotypes were revealed if human factor 1 (Ihu) was substituted for Imo: the order of the efficiency for the cleavage of C3(MA)hu was H.2 > H.1 = H.3. These results, taken together with the finding that the homologous combinations of mouse and human factors H and I expressed greater activity for the cleavage of C3(MA)hu than did the heterologous combinations of factor H and factor I, suggest that mouse factor H allotypes discriminate species of protease factor I but not those of substrate (C3(MA), and H.2 possesses the best compatibility for Ihu in C3(MA)hu inactivation.