Insertional mutants of the fungal maize pathogen Cochliobolus heterostrophus were screened for altered virulence. One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype. Analysis of sequence at the insertion site revealed a gene (CPS1) encoding a protein with two AMP-binding domains. CPS1 orthologs were detected in all Cochliobolus spp. examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants. Phylogenetic analysis suggested that CPS1 represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketidepeptide hybrids. Disruption of CPS1 caused reduced virulence of both race T and race O of C. heterostrophus on maize, of Cochliobolus victoriae on oats, and of Gibberella zeae on wheat. These results suggest that CPS1 functions as a general fungal virulence factor in plant pathogenic ascomycetes.