Amphotericin B associates with vesicles prepared from phosphatidylcholines. The influence of lipid composition on the initial rate of amphotericin B association with vesicles was examined using stopped-flow kinetic measurements. A relationship was found between the tightness of packing of phosphatidylcholine molecules in the vesicles and the initial rate of amphotericin B association. Shortening of the fatty acyl chain length of saturated phosphatidylcholines and increasing the number of double bonds in the fatty acyl chains of unsaturated phosphatidylcholines enhance the initial rate, whereas addition of cholesterol of the bilayers reduces the rate. The initial rate of association with phosphatidylcholine-sterol vesicles follows the order, thiocholesterol greater than androst-5-en-3beta-ol greater than epicholesterol greater than ergosterol greater than cholesterol and is thus inversely related to the order of phospholipid-sterol affinity, as revealed by permeability, surface area, and magnetic resonance measurements. These data suggest that the initial rate of amphotericin B uptake into vesicles depends on competition between lipid-lipid and amphotericin-lipid interactions.