The mitogen-activated protein kinase (MAPK) Mpk3/MpkC resembles the MAPK Hog1 but does not necessarily function in some filamentous fungi. Here, we compared functions of Mpk3 and Hog1 in Beauveria bassiana, a filamentous fungal insect pathogen, by multi-phenotypic analyses of their single/double deletion mutants. Growth defects of Delta mpk3 were moderate on all 14 minimal media with different carbon or nitrogen sources and less severe than those of Delta hog1 on most media tested. The double deletion mutant suffered significantly more severe growth defects than those observed in Delta mpk3 and Delta hog1, suggesting overlapping and collaborative roles of Mpk3 and Hog1 in uptake of six carbon and four nitrogen sources during normal growth. Despite little impact on conidiation capacity, mpk3 deletion slowed down conidial germination as much as hog1 or double deletion. Conidial resistance to UV-B irradiation decreased less in Delta mpk3 than in Delta hog1 or in the double mutant. The fungal virulence was similarly attenuated for all deletion mutants against Galleria mellonella larvae through normal cuticle infection. Intriguingly, the Delta mpk3 mutant displayed null response to high osmolarity and fludioxonil fungicide, to which both Delta hog1 and double mutants were hypersensitive and highly resistant, respectively, but it was more sensitive to a 3-h heat shock at 40 A degrees C than Delta hog1 during normal incubation. Western blot hybridization demonstrated that Mpk3 could collaborate with Hog1 in response to heat shock rather than to the chemical stresses. Altogether, Mpk3 collaborates with Hog1 only in response to heat shock and functions in sustaining the pest control potential of B. bassiana.