Microcrystalline cholesterol in either the anhydrous or monohydrate form was a potent activator of the alternative pathway of complement as measured by the electrophoretic conversion (crossed immunoelectrophoresis) of C3 and properdin factor B. Chelation with 0.01 M ethylene-diaminetetraacetate (EDTA) completely eliminated conversion, but 0.01 M ethyleneglycol tetraacetate (EGTA) had little or no effect. The magnitude of activation by cholesterol crystals was similar to that by zymosan, heat-aggregated IgG, or crystals of monosodium urate monohydrate. The microcrystalline forms of the acetate, linoleate, or oleate esters of cholesterol did not activate more complement than did saline controls. Cholestanol retained full C3 activating potency, but cholestane had none. Binding of IgG by cholesterol monohydrate is very small compared to that by sodium urate.