The putative specific interaction and complex formation by sphingomyelin and cholesterol was investigated. Accordingly, low contents (1 mol % each) of fluorescently labeled derivatives of these lipids, namely 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PyrPC), n-[10-(1-pyrenyl)decanoyl]sphingomyelin (PyrSM), and increasing concentrations of cholesterol (up to 5 mol %), were included in large unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), and the excimer/monomer fluorescence emission ratio (I(e)/I(m)) was measured. In DNPC below the main phase transition, the addition of up to 5 mol % cholesterol reduced I(e)/I(m) significantly. Except for this, cholesterol had only a negligible effect in both matrices and for both probes. We then compared the efficiency of resonance energy transfer from PyrPC and PyrSM to 22-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBDchol). An augmenting colocalization of the latter resonance energy transfer pair with temperature was observed in a DMPC matrix below the main phase transition. In contrast, compared to PyrSM the colocalization of PyrPC with NBDchol was more efficient in the longer DNPC matrix. These results could be confirmed using 5,6-dibromo-cholestan-3beta-ol as a collisional quencher for the pyrene-labeled lipids. The results indicate lack of a specific interaction between sphingomyelin and cholesterol, and further imply that hydrophobic mismatch between the lipid constituents could provide the driving force for the cosegregation of sphingomyelin and cholesterol in fluid phospholipid bilayers of thicknesses comparable to those found for biomembranes.