On the basis of mutational studies we have reported previously that residues Lys57, ArgSS, and Trp67 of human CRP contribute to the structure of the PCh-binding site. In this study we have compared the binding properties of four mutant CRPs K57Q/R58G, W67K, K57Q/R58G/W67K, and T76Y. All mutants were constructed by substituting SAP residues for the corresponding CRP residues. Wild-type (wt) and all mutant CRP cDNAs were expressed in COS cells and the recombinant proteins purified by affinity chromatography. K57Q/R58G/W67K and T76Y failed to bind solid-phase PCh-BSA. They did however bind with substantially reduced avidity to C-polysaccharide (PnC). W67K, K57Q/R58G/W67K, and T76Y CRP required 10-fold higher Ca2+ concentration than wt CRP to bind PnC and exhibited decreased affinities for mAb EA4.1, which recognizes a Ca2+ -dependent epitope. We conclude that Thr76 is a determinant of the PChbinding site probably interacting with the choline group. This conclusion is supported by recent crystallographic data which indicate that this residue participates in the formation of a hydrophobic pocket that constitutes the binding site for choline. Trp67 and to a lesser extent LysS7 and Arg58 appear to be reauired for the orooer conformation of the PCh-bindins site. but do not directly contact PCh.