Inactivation of human complement subcomponent C1-s by its regulator C1 inhibitor at physiological ionic strength proceeded at a 3-fold higher rate when C1-s was in the physiological C1- complex with subcomponents C1q and C1-r rather than as purified subunit. When the C1- complex was disassembled by chelation of calcium, the C1-s subcomponent was inactivated by C1 inhibitor at rates similar to those for the purified proteinase. Increasing ionic strength had little effect on the reaction of purified C1-s with C1 inhibitor but greatly diminished the rate of reaction of intact C1-. Addition of heparin accelerated the inactivation of purified C1-s by C1 inhibitor up to 25-fold but increased the inactivation of intact C1- only about 5-fold. These differences in the inactivation of C1-s by C1 inhibitor, depending on whether the proteinase is free or complexed with other subcomponents of C1-, suggest different mechanisms of reaction. Occurrence of subcomponent C1-s in a macromolecular complex with C1q and C1-r, thus, appears to be critical not only for directing its physiological activation but also its inactivation.