C4b-binding protein (C4BP) is a large complex assembly of eight subunits that functions as an inhibitor of the complement cascade. A portion of the C4BP in serum exists as a complex with protein S. This study demonstrated that another protein, serum amyloid P component (SAP), also formed a calcium-dependent complex with C4BP. The C4BP.SAP complex was detected by several methods including light scattering intensity, gel filtration, and sucrose density gradient ultracentrifugation. This complex was of high affinity relative to serum levels of these proteins so that no dissociation was detected at 3% of serum protein concentrations. The C4BP.SAP complex was also detected in normal serum and the results suggested that there was virtually no free SAP or uncomplexed C4BP in normal serum. In addition to its complex with C4BP, SAP underwent other calcium-dependent associations such as binding to phospholipid vesicles and self- aggregation. Self-aggregation was highly cooperative with kinetics corresponding to a reaction that was 6th-order with respect to calcium and required about 1.5 mM calcium. In contrast, formation of the SAP.C4BP complex and interaction of SAP with membranes required only about 0.4 and 1.0 mM calcium, respectively. Thus, selection of the correct conditions allowed study of the SAP.C4BP interaction without interference from self-aggregation. All three of these interactions of SAP were mutually exclusive and the SAP. C4BP interaction appeared to be favored over self-aggregation or binding of SAP to phospholipids. It seems likely that the biologically dominant interaction for SAP is with C4BP. The SAP.C4BP complex interacted with protein S and these binding sites appeared to be entirely independent. Furthermore, SAP had little or no effect on the ability of C4BP to bind C4b. Finally, the entire complex of proteins (C4BP, SAP, protein S, and C4b) could associate with membranes in the presence of calcium. Membrane binding occurred through the protein S component. This rather complicated assemblage of proteins probably functions in a regulatory role for the complement cascade or other biological systems. It is possible that elevated levels of SAP or nonequivalent levels of SAP and C4BP could contribute to certain pathological conditions.