To investigate the possibility that activation of the human alternative pathway of complement is influenced by cellular constituents that interact with C3b, the membrane proteins on rabbit and sheep erythrocytes that are associated with cell-bound human C3b have been analyzed. For both types of erythrocytes, activated C3b bound in a diffuse pattern via hydroxylamine-sensitive ester bonds. By using a homobifunctional crosslinker, a membrane component that has an apparent Mr of 35 kDa was shown to be noncovalently associated with C3b on sheep erythrocytes, but rabbit erythrocytes lacked a predominant C3b-associated protein. These studies suggest that regulation of human alternative pathway activity may be influenced by membrane glycoproteins that interact with cell-bound C3b.