The opposing actions of component and regulatory proteins of the alternative pathway of C on activation of the sequence were demonstrated in C2-deficient serum utilizing two initiating particles and incremental additions of proteins purified to homogeneity. Activation of the alternative pathway in C2-deficient serum by zymosan was assessed by measurement of the inactivation of C3 and B, while activation by rabbit erythrocytes (ER) was measured by their lysis. Participation of the classical C-component sequence was excluded by the presence of Mg-EGTA and by the failure of reconstitution of C2 to have a measurable effect. Incremental additions of either purified C3bINA or β1H, which increased the serum concentrations of the proteins by 43% to 175% or 15% to 30%, respectively, produced dose-related suppression of zymosan-induced B and C3 inactivation throughout the 45 min period of observation. Additions of either C3 or B to C2-deficient serum so as to increase their concentrations by 35% reversed the combined 50%-inhibitory effect of added C3bINA and β1H on the zymosan-induced inactivation of C3 and B. Dose-dependent inhibition of the rate and extent of lysis of ER in C2-deficient serum was obtained with increases of C3bINA concentrations of 25 to 100% and of β1H concentrations of 12.5 to 50%. As little as a 25% increase in the concentration of B increased the rate of lysis of ER in C2-deficient serum and partially reversed the inhibitory effect of the combined addition of C3bINA and β1H. Thus, the disequilibrium between component and control proteins of the alternative pathway created by the surface of an activating particle, that permits transition to the amplification phase of C3 cleavage, can be modulated by alterations in the concentrations of these proteins. Modest increases in the concentrations of C3bINA and β1H served to maintain balance, whereas comparable augmentations in the concentrations of C3 and B facilitated activation by the initiating particle.