Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase. Analysis by linear regression of the inhibitory effect of each procedure (r = 0.97, r = 0.98, respectively) for adjusting the number of heparin molecules/particle revealed that the dose-response relationships were identical and that complete inhibition occurred with greater than 12 X 10(8) molecules of heparin/zymosan particle. The coupling of heparin to zymosan did not impair the uptake of C3b from the fluid-phase interaction of C3, B, and D, and did not alter the capacity of bound C3b to associate with B so as to permit its inactivation by D. Although the regulatory proteins present in normal serum chelated with EDTA or presented as a combination of purified C3bINA and beta 1H were relatively inefficient in inactivating C3b function on an activating particle of the alternative pathway such as zymosan or zymosan-cyanogen bromide, the control proteins rapidly inactivated C3b on a nonactivating particle wuch as a sheep erythrocyte or zymosan with coupled heparin. The increased numbers of C3b sites susceptible to inactivation by C3bINA in the presence of beta 1H were significantly correlated to the number of molecules of heparin/particle. By linear regression analysis of the correlation (r = 0.99) the number of heparin molecules/particle required to promote total inactivation of bound C3b by purified control proteins was 13.8 X 10(6). This molecular analysis suggests that the action of heparin coupled to an activating particle of the alternative pathway is to promote the interaction between particle-bound C3b and the regulatory proteins, thereby preventing particle-associated amplified C3 cleavage. It is noteworthy that both surface constituents known to maintain a particle as a nonactivator of the alternative pathway, sialic acid and N-sulfated mucopolysaccharide, act by facilitating the inactivation by regulatory proteins of the function of particle-bound C3b.