We have studied the functions of the Trichoderma virens TmkB, a homologue of the yeast cell-wall integrity MAP kinase Slt2, using gene knockout. The functions of TmkB were compared to those of the pathogenicity MAP kinase homologue (TmkA). Like the tmkA loss-of-function mutants, tmkB mutants exhibited reduced radial growth and constitutive conidiation in dark as well as in liquid shake cultures. The tmkB mutants, in contrast to tmkA mutants, had cell-wall integrity defects, as shown by autolysis of the mycelia and increased sensitivity to cell-wall degrading enzymes. Interestingly, the tmkB mutants were not autolytic on the synthetic Vogels minimal medium. The tmkB mutants had attenuated ability to overgrow the plant pathogen Sclerotium rolfsii, while retaining the ability to overgrow Rhizoctonia solani and Pythium spp., a phenotype also exhibited by the tmkA mutants. This first functional analysis of a cell-wall integrity MAPK in Trichoderma spp., a group of economically important fungi, shows the importance of this signaling pathway in biocontrol. Common phenotypes of the TmkA and TmkB pathways suggest that the two MAPKs may share some substrates, perhaps subunits of key transcription factors, thus dependent on two phosphorylation events for their activity.