Because side effect profiles and key physical properties of liposomal amphotericin B reflect the molecular nature of the hydrated preparations, effort has been directed toward understanding this nature. We describe here an examination by differential scanning calorimetry in the region of the main transition of the phase behaviour of amphotericin B multilamellar liposomes used investigationally for patient treatment. Liposomes were composed of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (7:3 DMPC/DMPG) containing up to 33 mol% drug. Preparations in which pure DMPC or pure 1-oleoyl-2-stearoylphosphatidylcholine (OSPC) was substituted for 7:3 DMPC/DMPG were subjected to the same measurements for comparison. The DSC-derived partial phase diagrams were similar to those previously recorded using EPR spectroscopy for unsonicated liposomes of 7:3 DMPC/DMPG containing amphotericin B, and for mixtures with different pure saturated and unsaturated phosphatidylcholines (Grant, C.W.M., et al. (1989) Biochim. Biophys. Acta 984, 11-20). Fluidization onset temperatures for liposome host matrices were relatively unaffected by drug compared to the temperatures of completion. This effect was particularly marked for the unsaturated phospholipid matrix. Partial phase diagrams were interpreted as demonstrating that amphotericin B has a tendency to separate into a rigid phase within the membrane. This is consistent with molecular modelling considerations which suggest that amphotericin B may exist as oligomers in a phospholipid matrix. Drug-induced alterations of DSC melting profiles for the phospholipid bilayers studied were less extensive than those reported for partially sonicated preparations of 7:3 DMPC/DMPG (Janoff, A.S., et al. (1989) Proc. Natl. Acad. Sci. USA 85, 6122-6126). Melting profiles obtained did not change upon further sample incubation, suggesting that the hydrated preparation represented a thermodynamically stable form.