HAS cell cholesterol level modulates fusion activity. Double-labeled RBC (DiI C-18 and carboxy fluorescein, CF) were bound to HAS and GPI-HAS cells, and fusion triggered by application of a pH 4.9 solution for 2 min at 30°C. Cells were then incubated with pH 6.4 impermeant buffer, and the percentage of bound cells showing lipid dye transfer and cytoplasmic dye transfer were counted in a fluorescence microscope. Cholesterol and oleic acid enrichments were achieved as described in Materials and methods. Cholesterol mimics oleic acid in modulating fusion of HAS cells at the pH optima but not at pH 5.3. Cholesterol did not modulate fusion of GPI-HAS.