Membrane cofactor protein (MCP) of the C system is a widely distributed regulatory molecule with C3b/C4b binding and factor I-dependent cofactor activity. A rabbit polyclonal antibody was raised against purified human MCP, and it was found to also immunoprecipitate C4b-binding protein (C4bp). Other related complement regulatory proteins, factor H, C3b/C4b receptor, and decay-accelerating factor, were not recognized by this polyclonal antibody to MCP. The cross-reactive epitope was sensitive to reduction with 2-ME and about 3% of the anti-MCP antibody reacted with C4bp. The amino-terminal 48,000-Da, chymotryptic fragment of C4bp was recognized by the antibody to MCP. This fragment of C4bp contains a seven-amino acid peptide that is identical, in its sequence and its location in the third short consensus repeat, to one found in MCP. Two polyclonal antibodies to C4bp, one raised to native and the other to reduced C4bp, did not cross-react with MCP. In addition to this one-way cross-reaction with C4bp, a protein with a m.w. of approximately 60,000 (p60) was found in two of three C4bp preparations that also cross-reacted with antiserum to MCP. p60 was present in trace quantities in the C4bp preparation and was successfully isolated from plasma by C3b affinity chromatography. Its Mr was distinct from that of MCP and other known C3b/C4b binding proteins. Furthermore, p60 was isolated by two different procedures and such material possessed no detectable cofactor activity. Based on these results, p60 is a plasma C3b-binding protein that shares epitopes with C4bp and MCP, and is probably not a soluble form of MCP.