Amphotericin B (AmB) is a lifesaving polyene antibiotic used widely to treat deep-seated mycoses. Both the pharmaceutical effectiveness as well as toxic side effects depend on molecular organization of the drug. In the present study, we analyzed steady-state fluorescence, fluorescence anisotropy spectra, fluorescence lifetimes, and fluorescence anisotropy decays of AmB in the systems believed to ensure monomeric organization of the drug and in model lipid membranes. The results of the analyses show that in all of the systems studied, the drug appears in, at least, two spectral forms, interpreted as monomeric and aggregated. Spectroscopic and fluorescence lifetime characteristics of both forms are provided. Interpretation of the fluorescence anisotropy spectra of AmB incorporated into liposomes formed with dipalmitoylphosphatidylcholine let us conclude that monomers of the drug are more tightly bound to the lipid membranes as compared to the aggregates and that AmB aggregates destabilize the membrane structure. Structural model analysis, compared to the analysis of spectral shifts, leads to the conclusion that basic constituents of AmB aggregated structure is a tetramer composed of two hydrogen-bond-stabilized dimers, each dimer formed by molecules twisted by ca. 170°. The tetramer itself can span lipid bilayers and can act as a transmembrane ion channel. Specific aggregate formation of AmB has been concluded as a universal and ubiquitous form of molecular organization of the drug. This process is discussed in terms of toxic side effects of AmB.