Fluorescence and infrared spectroscopy and cholesterol oxidase activity were employed to investigate the effect of phosphandylcholine (PC) acyl chain length mismatch on the lateral organizations of lipids in liquid-ordered dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/CHOL) bilayers. Plots of steady-state fluorescence emission anisotropy of diphenylhexatriene (DPH) labeled PC (DPH-PC) embedded in the DPPC/DLPC/CHOL bilayers revealed significant peaks at several DPPC mole fractions (Y-DPPC) when the cholesterol mole fraction (X-CHOL) was fixed to particular values Analogously, the DPH-PC anisotropy peaked at several critical X-CHOL's when Y-DPPC was fixed Acyl chain C-H and C=O vibrational peak frequencies of native PC as well as the activity of cholesterol oxidase also revealed dips and peaks at similar Y-DPPC's Importantly, most of the observed peaks/dips coincide with the critical mole fractions predicted by the Superlattice (SL) model A three-dimensional map of DPH-PC anisotropy versus composition in the range 0.32 <= X-CHOL <= 50; 0 54 <= Y-DPC <= 0.72 revealed a prominent peak at (X-CHOL, Y-DPPC) approximate to (0.42, 0 64). This suggests a simultaneous presence of two different types of superlattices, one where cholesterol is the quest molecule in a PC host lattice and another where DPPC is the guest in the DLPC host lattice Time-resolved measurements of DPH-PC fluorescence indicated the existence of an ordered, rotationally hindered environment of acyl chains at that "critical" composition consistent with the existence of SL arrangements. We propose that beside CHOL/PC superlattices, DPPC, and DLPC as well tend to adopt regular SL-like lateral distributions relative to each other, presumably because the less hydrophobic DLPC molecule is slightly displaced toward the aqueous phase, thus allowing more room and mobility for the head groups of both DPPC and DLPC as well as for the acyl chain tails of DPPC The parallel presence of two kinds of superlattices, that is, CHOL/PC-SL and DPPC/DLPC-SL as demonstrated here, has intriguing implications regarding lipid homeostasis of eukaryote membranes