Pyrrolnitrin, produced by several bacteria that are used in biological control, has an inhibitory effect on the electron transport system of respiration in Neurospora crassa. We have previously described that fludioxonil, a derivative of phenylpyrroles, affects osmotic signal transduction. Both pyrrolnitrin and fludioxonil were highly active against Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, and N. crassa. However, a high concentration of pyrrolnitrin (more than 10 μg/ml) inhibited the growth of fludioxonil-insensitive fungi such as Pythium ultimum, Phytophthora capsici, and Saccharomyces cerevisiae. In order to clarify the difference in the antifungal mechanisms between pyrrolnitrin and fludioxonil, we observed cross-resistance in mutants of the osmotic signal transduction pathway, namely, os-1 (histidine kinase), os-4 (MAPKK kinase), os-5 (MAPK kinase), and os-2 (MAP kinase) of N. crassa. All os mutants that were resistant to fludioxonil showed cross-resistance to pyrrolnitrin without exception. The levels of resistance to pyrrolnitrin correlated well with those to fludioxonil in the 10 os-1 mutant alleles with single amino acid substitutions. However, at a concentration of 6.1 μg/ml, pyrrolnitrin inhibited the growth of all strains including the os mutants insensitive to fludioxonil even at 25 μg/ml. When the conidia of the wild-type strain were grown on a medium containing either fungicide at a concentration of 0.1 μg/ml, both fungicides induced the swelling and rupture of conidia without germ-tube formation. At a concentration of 25 μg/ml, pyrrolnitrin inhibited conidia germination without any morphological change in the fludioxonil-insensitive os-S mutant. Both fungicides at a concentration of 1 μg/ml stimulated glycerol synthesis in the wild-type strain, but the glycerol content was reduced to a considerable extent on treatment with 25 μg/ml pyrrolnitrin. These results suggest that a primary antifungal mechanism of pyrrolnitrin against N. crassa is interference with the osmotic signal transduction pathway rather than inhibition of respiration.