In vitro effects of the polyene antibiotic amphotericin B (AmB) on growth, viability, and ultrastructure of amoeboflagellates of the genus Naegleria were examined. The strains studied were the nonpathogenic Naegleria gruberi EG(B) and the Carter and TY strains of the pathogenic Naegleria fowleri. AmB was amoebicidal at all concentrations used (0.25, 0.50, and 1.0 mug/ml) when the drug was added to cultures in lag phase, as determined by viability testing, but was mainly inhibitory when added to log-phase cultures. The drug produced ultrastructural modifications at all concentrations (0.05 to 1.0 mug/ml). These changes included distortion of nuclear shape, increase in cytoplasmic membranes (both rough and smooth endoplasmic reticulum), decrease in number of food vacuoles, absence of pseudopod formation, mitochondrial abnormalities, increase in autophagic vacuoles, and blebbing of the plasma membrane. These alterations of amoebic ultrastructure became more pronounced with increased time in AmB and with increase in AmB concentration in the growth medium.