Serum depleted of the first component of complement (C1D) was prepared by treating fresh human serum with Sepharose-IgG in the presence of triethylenetetramine-N,N,N',N'',N''',N'''-hexa-acetic acid and di-isopropyl fluorophosphate at acidic pH (5.2). The total hemolytic activity of this C1D could be increased to approximately 75% of that of the original serum by the addition of excess purified C1. A linear relation was obtained on determination of C1 hemolytic activity using C1D, and the C1 titers of sera from patients measured by this simple method showed a good correlation with those measured using intermediate cells.