A new immunochemical method was used to quantitate the number of C3 molecules bound to human red cells in vitro or in vivo and to assess the clinical significance of cell-bound C3 in immune hemolysis. In addition, results utilizing the immunochemical method were compared with those obtained using commonly performed semiquantitative serologic techniques such as the antiglobulin test and antiglobulin titration score. The antiglobulin test using anti-C3 antiglobulin serum became weakly positive with 60-115 molecules C3 per red cell, and was strongly positive with 1000 molecules C3 per red cell. Antiglobulin titration scores correlate well with immunochemical assessment of the number of C3 molecules per red cell. Therefore, a simple extension of the routine antiglobulin test affords clinically useful data concerning the relative degree of sensitization of red cells by C3. Two of fourteen patients with fewer than 1100 molecules C3 per red cell had hemolytic anemia, whereas 8 of 11 patients with greater than 1100 molecules C3 per red cell had overt hemolysis. The presence or absence of hemolysis was not explained by variations in the amount of IgG on these patients’ RBC. It thus appears that the amount of C3 per red cell is an important determinant of hemolysis in human immune hemolytic anemias.