The lectin pathway of complement is an important effector arm of innate immunity. It forms a first line of defense against invading pathogens and dangerously altered self structures. Pattern recognition molecules (mannose-binding lectin (MBL), ficolins) bind to the dangerous particles, which is followed by activation of MBL-associated serine proteases, MASP-1 and MASP-2, resulting in the initiation of the complement cascade. The activation of the lectin pathway is strictly controlled by natural inhibitors, since uncontrolled activation can lead to serious self-tissue damage. Recently we have shown that inhibition of either MASP-1 or MASP-2 by in vitro evolved specific inhibitors completely blocks the lectin pathway in human serum. In this study, we examined the inhibitory action of C1-inhibitor (C1-inh), antithrombin (AT) and α2-macroglobulin (α2M) on MASP-1 and MASP-2, and studied the inhibition of the lectin pathway in normal human serum in the presence and absence of heparin using C3 and C4 deposition assays. We measured the association rate constants for the serpin/protease reactions. We found that in the presence of heparin both C1-inh and AT are equally efficient inhibitors of the lectin pathway. Although α2M formed complex with MASP-1 in fluid phase, it could not abolish lectin pathway activation on activator surfaces.