In the past decade substantial progress has been made in understanding the organization and biological activity of amphotericin B (AmB) in the presence of sterols in lipid environments. This review concentrates mainly on interactions of AmB with lipids and sterols, AmB channel formation in membranes, AmB aggregation, AmB modifications important for understanding its biological activity, and AmB models explaining its mechanism of action. Most of the reviewed studies concern monolayers at the water-gas interface, monolayers deposited on a solid substrate by use of the Langmuir-Blodgett technique, micelles, vesicles, and multi-bilayers. Liposomal AmB formulations and drug delivery are intentionally omitted, because several reviews dedicated to this subject are already available.