ABSTRACT Acremonium chrysogenum , the fungal producer of the pharmaceutically relevant β-lactam antibiotic cephalosporin C, is classified as asexual because no direct observation of mating or meiosis has yet been reported. To assess the potential of A. chrysogenum for sexual reproduction, we screened an expressed sequence tag library from A. chrysogenum for the expression of mating type ( MAT ) genes, which are the key regulators of sexual reproduction. We identified two putative mating type genes that are homologues of the α-box domain gene, MAT1-1-1 and MAT1-1-2 , encoding an HPG domain protein defined by the presence of the three invariant amino acids histidine, proline, and glycine. In addition, cDNAs encoding a putative pheromone receptor and pheromone-processing enzymes, as well as components of a pheromone response pathway, were found. Moreover, the entire A. chrysogenum MAT1-1 (Ac MAT1-1 ) gene and regions flanking the MAT region were obtained from a genomic cosmid library, and sequence analysis revealed that in addition to Ac MAT1-1-1 and Ac MAT1-1-2 , the Ac MAT1-1 locus comprises a third mating type gene, Ac MAT1-1-3 , encoding a high-mobility-group domain protein. The α-box domain sequence of Ac MAT1-1-1 was used to determine the phylogenetic relationships of A. chrysogenum to other ascomycetes. To determine the functionality of the Ac MAT1-1 locus, the entire MAT locus was transferred into a MAT deletion strain of the heterothallic ascomycete Podospora anserina (the PaΔMAT strain). After fertilization with a P. anserina MAT1-2 (MAT + ) strain, the corresponding transformants developed fruiting bodies with mature ascospores. Thus, the results of our functional analysis of the Ac MAT1-1 locus provide strong evidence to hypothesize a sexual cycle in A. chrysogenum .