The fungicidal activity of six rabbit neutrophil cationic peptides (NP) against resting (dormant) spores, preincubated (swollen) spores, and hyphae of Aspergillus fumigatus and Rhizopus oryzae was examined. Whereas resting spores of both fungi were totally resistant to killing by up to 500 μg of NP/ml, preincubated spores and hyphae were sensitive, under optimal conditions, to <1 μg/rnl. NP-1 and NP-2, also found in rabbit bronchoalveolar macrophages, were the most active of the peptides. Activity was inhibited with increasing ionic strength, calcium but not magnesium, incubation at 4 C, and the chitin fragments chitobiose and chitotriose. ¹²⁵I-labeled NP-1 bound to all three forms of A. fumgatus. Finally, killing of A. fumigatus conidia by rabbit bronchoalveolar macrophages was faster and greater if the conidia werefirst preincubated in RPMI 1640 medium. Thus, although NP did not kill the resting A. fumigatus conidia, a role for cationic peptides in macrophage-mediated killing is not precluded, as fungicidal activity of both isolated cationic peptides and intact bronchoalveolar macrophages appeared to occur only after the conidia became activated.