The relationship between the molecular organization of lipid headgroups and the activity of surface-acting enzyme was examined using a bacterial cholesterol oxidase (COD) as a model. The initial rate of cholesterol oxidation by COD in fluid state 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers was measured as a function of POPE-to-phospholipid mole ratio (XPE) and cholesterol-to-lipid mole ratio (XCHOL) at 37 °C. At XPE = 0, the COD activity changed abruptly at XCHOL ≈ 0.40, whereas major activity peaks were detected at XPE ≈ 0.18, 0.32, 0.50, 0.64, and 0.73 when XCHOL was fixed to 0.33 or 0.40. At a fixed XCHOL of 0.50, the COD activity increased progressively with PE content and exhibited small peaks or kinks at XPE ≈ 0.40, 0.50, 0.58, 0.69, and 0.81. When XPE and XCHOL were systematically varied within a narrow 2-D lipid composition window, an onset of COD activity at XCHOL ≈ 0.40 and the elimination of the activity peak at XPE ≈ 0.64 for XCHOL >0.40 were clearly observed. Except for XPE ≈ 0.40 and 0.58, the observed critical PE mole ratios agree closely (±0.03) with those predicted by a headgroup superlattice model (Virtanen, J.A., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4964−4969; Cannon, B., et al. (2006) J. Phys. Chem. B 110, 6339−6350), which proposes that lipids with headgroups of different sizes tend to adopt regular, superlattice-like distributions at discrete and predictable compositions in fluid lipid bilayers. Our results indicate that headgroup superlattice domains exist in lipid bilayers and that they may play a crucial role in modulating the activity of enzymes acting on the cell membrane surface.