The alternative or properdin pathway of complement is primarily controlled by the endopeptidase C3b inactivator (C3bINA) and the nonproteolytic glycoprotein β1H. The molecular mechanisms of control were investigated by performing binding studies of radiolabeled complement proteins to C3b bearing sheep erythrocytes (E_SC3b). C3b was found to have distinct binding sites for β1H, C3bINA, Factor B, and properdin. β1H binding increased C3bINA binding 30-fold, while Factor B binding prevented C3bINA action on C3b and was competitive with β1H binding. Properdin binding, which facilitates Factor B interaction with C3b, had no effect on the β1H and C3bINA sites. Activators such as rabbit erythrocytes (E_R) have previously been shown to interfere with the effectiveness of the control by C3bINA and β1H, thereby allowing unrestricted formation of C3 convertase. Such restriction of control does not occur on the surface of E_S, a nonactivator of the alternative pathway. On the basis of comparative binding studies, restriction of control is explained entirely by reduced binding of β1H to E_RC3b relative to E_SC3b. Access of properdin, Factor B, C3bINA, and the Fab fragment of anti-C3 to the two cell types was unrestricted. Restriction of β1H control could be generated on the surface of E_S by removal of cell-surface sialic acid with neuraminidase (acylneuraminyl hydrolase; EC 3.2.1.18). This enzymatic treatment converted E_S from a nonactivator to an activator of the alternative pathway.