Abstract A method is described for the determination of the absolute amount of guinea pig (GP) C3 bound to cell surfaces. The test is based on the inhibition of anti-C3 by C3. The C3 to be measured is added to a standard amount of anti-C3 antibody and the residual activity is determined by its capacity to sensitize EC43gp for lysis by GPC. By comparing the observed lysis with inhibition-of-lysis curves obtained with known C3 antigen, the amount of test antigen can be determined. The method is simple, rapid, and reproducible and can detect nanogram amounts of C3. The C3 content of six normal guinea pig sera was found to be 1365 ± 56 µg/ml (ranges 1120 to 1510 µg/ml) by the inhibition method described in this paper. Guinea pig hepatoma cells, line-1 and line-10, harvested from the peritoneal cavity of guinea pigs contained approximately 6.7 × 103 to 6.2 × 104 GPC3 per cell whereas tumor cells sensitized with anti-Forssman or specific anti-tumor antibody contained from 1 to 9 × 105 C3 per cell. Susceptibility to killing by antibody and GPC could not be correlated with the amount of GPC3 bound to the surfaces of the tumor cells. Comparisons made between the amounts of C3 bound to antibody-sensitized sheep erythrocytes and nucleated cells suggest there is a fundamental quantitative difference in the fixation of selected C components.