Clinical studies have shown that moderate alcohol consumption protects from atherosclerosis by altering cholesterol levels, but the mechanisms of ethanol-cholesterol effects on the membrane remain unknown. To study the effect of ethanol on neutrophils with low or high cholesterol content, we treated neutrophils with MßCD for 35% depletion or with MßCD-cholesterol complexes for 40% enrichment, which decreased or increased membrane fluidity, respectively. Neutrophils were incubated with physiologically relevant ethanol concentrations achieved in humans by alcohol consumption (<0.5% ethanol by vol.), washed and perfused over parallel-plate flow chambers with P-selectin-coated 1-µm-diameter beads or P-selectin surfaces at a venous shear rate of 100 s-1. Cholesterol enrichment increased the tethering fraction of neutrophils from 0.63 to 0.74, while cholesterol depletion decreased it to 0.45. Tether length and lifetimes also increased from 2.50 to 5.30 µm and 0.53 to 0.62 sec with cholesterol enrichment, while cholesterol depletion reduced them to 0.67 µm and 0.38 sec. Tether growth velocity doubled with cholesterol enrichment. The fraction of firm arrest increased from 0.05 to 0.22, and cholesterol-treated cells were 13% larger in size and more deformable, suggesting that high-cholesterol cells may be more susceptible to activation. Treatment with ethanol mitigated these cholesterol-induced effects, but the effect of ethanol on cholesterol-treated cells was significantly smaller than its effect on neutrophils with basal cholesterol level. Ethanol also reduced CD11b expression and caused PSGL-1 redistribution, which may contribute to the change in neutrophil adhesive behavior. Our results indicate that both ethanol and cholesterol affect bond mechanics, membrane fluidity, and surface molecules to change neutrophil response during receptor-mediated adhesion.