C1-inhibitor is increasingly used experimentally and clinically in inflammatory conditions like septicemia and ischemia-reperfusion injury. Several mechanisms may account for the anti-inflammatory effects of C1-inhibitor, including inhibition of complement. The aim of the present study was to investigate and compare the supraphysiologic effect of C1-inhibitor on the three complement pathways. Novel assays for specific evaluation of the classical, lectin and alternative pathways were employed using normal human serum supplemented with increasing concentrations of C1-inhibitor. Solid-phase classical- and lectin pathway activation was dose-dependently and significantly reduced up to 85% in the range of 2-28 times physiologic C1-inhibitor concentration. The lectin pathway was more potently inhibited than the classical at low doses. A functional lectin pathway assay demonstrated a significant reduction of C4 deposition up to 86% even at low concentration of C1-inhibitor and documented the effect to be at the level of MBL/MASPs. In contrast, C1-inhibitor had no effect on solid-phase alternative pathway activation, but significantly reduced cobra venom factor-induced fluid-phase activation up to 88%. The negative controls albumin and IgG had no effect on complement activation. The positive inhibitory controls compstatin (C3 inhibition), EDTA- or MBL-deficient sera reduced complement activation by 82-100%. We conclude that C1-inhibitor in high physiologic doses differentially inhibits all three-complement pathways. The inhibition pattern was strikingly different in the classical and lectin pathway, compared to the alternative. Previous studies interpreting the effects of C1-inhibitor as only due to classical pathway inhibition needs reconsideration. The data has implications for the therapeutic use of C1-inhibitor.