The kinetics of formation of amphotericin B (AmB) aqueous pores in ergosterol-containing DMPC or egg-PC liposomes was investigated using a stopped-flow method. The formation of aqueous pores by AmB occurred very rapidly (in milliseconds to seconds depending of the AmB concentration), and it was always preceded by the formation of transient, non-aqueous pre-pore structures. As anticipated, these non-aqueous pre-pore structures made the liposomes more permeable to urea without at the same time leading to a decrease of the reflection coefficient of urea or to an enhancement of glucose permeability. However, when liposomes were composed of egg-PC and cholesterol, the formation of non-aqueous and aqueous channels by AmB occurred after a lag time of several minutes. Such a time lag for AmB action was not observed in cholesterol-containing DMPC liposomes, an indication that the phospholipid composition is an important parameter in the formation of non-aqueous channels by AmB. Both non-aqueous and aqueous channels were always formed at lower concentrations of AmB in liposomes containing ergosterol while higher concentrations were needed in cholesterol-containing liposomes. Measurements of the permeabilizing effect of AmB on liposomes prepared without sterols indicate that non-aqueous channels were formed in DMPC (but not in egg-PC) at polyene concentrations identical to that found for cholesterol-containing liposomes. No evidence of the formation of aqueous channels by AmB was found in pure DMPC liposomes. These data are consistent with the concept that AmB forms non-aqueous channels without the direct participation of sterol molecules. The initially formed non-aqueous channels subsequently interact with the sterols in the membrane to form aqueous channels, having an enlarged diameter. This sequential mechanism for the formation of AmB aqueous pores in liposomes provides a rationale for the understanding of the effect of both the phospholipid composition and type of sterol in the interaction of AmB with natural membranes and artificial bilayers.