The interaction of rabbit C-reactive protein (rCRP) with a supported monolayer containing a phosphorylcholine moiety was studied. Three types of phospholipids were synthesized, each containing a insertion spacer of eight, six, or three atoms between the phosphorylcholine group and hydrophobic tail. By varying the length of the insertion spacer, we can vary the extension of the phosphorylcholine group from the membrane surface. By varying the monolayer composition, we can control the lateral distance between the exposed phosphorylcholine groups. Using the surface plasmon resonance technique (SPR), we demonstrated that the calcium-dependent binding of rCRP to the model membrane is governed not only by the ability of the ligand to access the binding pocket fully (spacer length), but also by lateral hindrance within the two-dimensional plane of the membrane. The value of the apparent binding constant was estimated by theoretical analysis, which is obviously dependent on the composition of the lipid mixture, and a maximum of (9.9 6 1.5) 3 106 M21 was obtained.