A simple one-step procedure has been developed for the molecular titration of C2 by utilizing the ability of the test material to restore the hemolytic activity of human serum selectively deficient in C2 (C2D serum). In this assay, equal volumes of EA (10⁸ cells/ml), C2D serum (1/20), and a suitable dilution of a source of C2 were incubated at 37°C for 60 min and the fraction of cells lysed was used to calculate the effective molecules of C2/ml test material. The assay can be used to titrate C2 in human, guinea pig, rat, mouse and rabbit sera, but not C2 in dog serum. The assay is simple and reproducible and comparable in sensitivity to the conventional two-step assay with EAC14 cells and C^gp-EDTA.