The molecular action of amphotericin B (AmB) on the cell membranes of both AmB-susceptible and AmB-resistant fungal cells was investigated through the use of the fluorescent membrane probe trimethylammonium diphenylhexatriene (TMA-DPH). AmB, the most effective drug for the treatment of systemic fungal infections, is known to interact specifically with membrane sterols, especially ergosterol (the major sterol in fungal cells). Treatment of AmB-susceptible Candida albicans and Cryptococcus neoformans cells with AmB induced a novel biphasic change in TMA-DPH fluorescence intensity over time. The initial decrease in fluorescence intensity results from energy transfer between AmB and TMA-DPH when AmB binds to the fungal cell membrane. The second phase of increasing fluorescence intensity is interpreted in terms of a combination of probe repartitioning and probe segregation as a result of the formation of membrane pores via the aggregation of AmB-ergosterol complexes. An AmB-resistant strain of C. neoformans, containing 94% of aberrant delta-8 double-bonded ergosterol precursors and only 4% of ergosterol (74% ergosterol in wild-type cells), exhibited the first phase of AmB binding but not the second phase of increasing fluorescence intensity. This result suggests that AmB's antifungal activity lies in its ability to form membrane pores due to aggregation of AmB-ergosterol complexes. The AmB-Induced biphasic fluorescence intensity profile may lead to further elucidation of the molecular action of AmB on fungal cells and may provide a sensitive method for screening the development of drug resistance in fungal cells.