Langmuir surface pressure, small-angle neutron scattering (SANS), and neutron reflectivity (NR) studies have been performed to determine how formulation of the antifungal drug amphotericin B (AmB), With sodium cholesteryl sulfate (SCS)-as in Amphotec-affects its interactions with ergosterol-containing (model fungal cell) and cholesterol-containing (model mammalian cell) Membranes. The effects of Mixing AmB in 1:1 molar ratio with cholesteryl sulfate (yielding AmB-SCS micelles) are compared against those of free AmB, using monolayers and bilayers formed from palmitoyloleoylphosphatidylcholine (POPC) in the absence and presence of 30 mol % ergosterol or cholesterol, in all cases employing a 1:0.05 molar ratio of lipid:AmB Analyses of the (bilayer) SANS and (monolayer) NR data indicate that the equilibrium changes in membrane structure induced in sterol-free :and sterol-containing membranes are the same for free AmB and AmB-SCS. Stopped-flow SANS experiments, however, reveal that the structural changes to vesicle membranes occur far more rapidly following exposure to AmB-SCS vs free drug, with the kinetics of these changes varying with membrane composition. With POPC vesicles, the structural changes induced by AmB-SCS become apparent only after several minutes, and equilibrium is reached after similar to 30 min The corresponding onset of changes in POPC-ergosterol and POPC-cholesterol vesicles, however, occurs within similar to 5 s, with equilibrium reached after 10 and 120 s, respectively. The rate of insertion of AmB into POPC-sterol membranes is thus increased through formulation as AmB-SCS: Moreover, the differences in monolayer surface pressure and SANS structure-change equilibration times suggest significant rearrangement of AmB within these membranes following insertion. The reduced times to equilibrium for the POPC-ergosterol vs POPC-cholesterol systems are consistent with the known differences in affinity of AmB for these two sterols, and the reduced time to equilibrium for AmB-SCS interaction with POPC-ergosterol membranes vs that for free AmB is consistent with the reduced host toxicity of Amphotec.