Membrane cofactor protein (MCP) (CD46) of the C system binds to C3b and C4b, functions as a cofactor for their cleavage, and protects autologous cells from C-mediated injury. The predominant structural motif of MCP is the short consensus repeat (SCR), a repeating domain involved in ligand binding of other related C regulatory proteins. SCR deletion mutants were constructed to determine which of the four SCR of MCP contribute to ligand binding and cofactor activity. ELISA were developed to evaluate binding efficiency of mutants to ligand. Analysis of the deletion mutants indicated that the third and fourth SCR were important for both ligand binding and cofactor activity of C3b (iC3) and C4b. In addition, the same SCR were required for efficient binding of an mAb known to inhibit MCP function. The mutant deleted of SCR-2 bound but lacked cofactor activity for iC3. It did not bind or possess cofactor activity for C4b. Deletion of the first (amino-terminal) SCR had a minimal effect on iC3 binding and cofactor activity but reduced the efficiency of C4b binding. The results identify the SCR of MCP that contribute to ligand binding and cofactor activity. The data also suggest the presence of distinguishable iC3 and C4b binding sites and provide evidence that iC3 binding is not always sufficient for cofactor activity.