Small unilamellar vesicles composed of phosphatidylserine (PS) and cholesterol at various ratios were employed in studying the effect of cholesterol on Ca2+-induced vesicle aggregation and fusion using the Tb/dipicolinic acid assay. The leakage of preencapsulated Tb3+ was also measured. The analysis of the data provided estimates for the rate of aggregation C11, and the rate of fusion per se, f11. An increase in cholesterol contents results in a decrease in C11 values. Similarly, aggregation of PS/cholesterol vesicles is slower than that of PS vesicles in the presence of 650 mM NaCl. With 100 or 200 mM NaCl, the overall fusion rate of PS/cholesterol vesicles is slower than that of PS vesicles; the rate being reduced by an increase in cholesterol contents. With 600 mM NaCl, the overall fusion rate of PS/cholesterol 9:1 vesicles is faster than that of PS vesicles, and results are well-simulated by assuming no delay in vesicle aggregation up to dimers. Emerging f11 values are larger in PS/cholesterol than in PS vesicles. An analysis of fusion kinetics of several lipid concentrations shows that f11 values of PS/cholesterol 3:1 vesicles are 5-times larger than those of PS vesicles, when fusion occurs in a medium containing 200 mM NaCl and 1.5 mM Ca2+. The increase in Na+ concentration from 100 to 200 mM, or 600 mM results in a 50- or 150-fold reduction in f11 values of PS vesicles. It is suggested that incorporation of cholesterol in PS vesicles results in enhancement of Ca2+-induced fusogenic capacity.