Members of the fungal kingdom serve as models for numerous cellular processes, among them sexuality.1 In heterothallic ascomycetes, mating-type systems ensure that only compatible isolates fuse to enter the sexual phase.2,3,4,5,6 This includes reciprocal secretion and recognition of pheromones, commonly termed α-factor and a-factor, which are processed from peptide precursors.7,8,9,10 Identification of fungal mating pheromones and their cognate receptors has been achieved by homology searches11,12,13,14,15,16,17; however, this approach had failed to detect a-factor-like pheromones from Eurotiomycetes,5,18,19,20,21 a fungal group including medically and economically important species.22 Sexuality of the opportunistic pathogen Aspergillus fumigatus23,24,25 is genetically determined by a bipolar mating-type system encoding MAT1-1-1 and MAT1-2-1 regulators.16,26,27,28,29,30 By analyzing transcriptome data from strains overexpressing the corresponding MAT genes,31 we identified a candidate pheromone precursor gene B (ppgB) to encode the elusive Eurotiomycete a-factor pheromone. Its deduced peptide is 24 aa in length and features a canonical CaaX farnesylation motif. Further analyses provided supporting evidence that PpgB is a prototype for the a-factor-like pheromone of the aspergilli, including expression of ppgB in a MAT1-2-1-dependent manner, and that an A. fumigatus ppgBΔ deletion strain was unable to mate and form fruiting bodies with a compatible partner. Inspection of Aspergillus genomes from members of the section Fumigati revealed high conservation of PpgB sequence as well as of the α-factor-like PpgA, indicating that incompatibility factors other than solely pheromone discrimination are responsible for speciation. The identification of the A. fumigatusa-factor-like pheromone closes a substantial knowledge gap with respect to cellular recognition and sexual propagation of Eurotiomycete fungi.