A hemolytic plate assay specific for active human complement component C3 is described. The method is well suited for tracing active C3 during preparative purification or for screening of plasma samples. The assay is based on activation of the alternative pathway of complement by unmodified rabbit erythrocytes. Plasma treated with methylamine supplies the essential complement components other than C3. The lytic reaction is complete in 5 h at 37 degrees C and is unchanged by incubation overnight. The dose-response curve, i.e., lysis diameter versus logarithm of C3 concentration, is linear within 0.1-10 times normal plasma concentrations of C3. The standard deviation is below 10%. The hemolytic agarose plates are easy and inexpensive to prepare, and they can be stored at 4 degrees C for 2 weeks before use. This paper describes the optimal conditions of the assay and proves its specificity. Its use in C3 preparation and plasma screening for C3 is discussed.