Technical Abstract: In heterothallic ascomycete fungi, idiomorphic alleles at the MAT locus control two sex pheromone/receptor pairs that function in recognition and chemoattraction of strains with opposite mating types. In the ascomycete Gibberella zeae, the MAT locus is rearranged such that both alleles are adjacent on the same chromosome. Strains of G. zeae are self-fertile, but they can outcross facultatively. Our objective was to determine if pheromones retain a role in sexual reproduction in this homothallic fungus. Putative pheromone precursor genes (ppg1 and ppg2) and their corresponding pheromone receptor genes (pre2 and pre1) were identified in the genomic sequence of G. zeae by sequence similarity and microsynteny with other ascomycetes. ppg1, a homolog of the Saccharomyces a-factor pheromone precursor gene, was expressed in germinating conidia and mature ascospores. Expression of ppg2, a homolog of the a-factor pheromone precursor gene, was not detected in any cells. pre2 was expressed in all cells, but pre1 was expressed weakly and only in mature ascospores. Deletion mutations delta ppg1 or delta pre2 reduced fertility in self-fertilization tests by approximately 70%. Delta ppg1 reduced male fertility and delta pre2 reduce female fertility in outcrossing tests. In contrast, delta ppg2 and delta pre1 had no discernable effects on sexual function. Delta ppg1/ delta ppg2 and delta pre1/delta pre2 double mutants had the same phenotype as the delta ppg1 or delta pre2 single mutants. Thus, one of the putative pheromone/receptor pairs (ppg1/pre2) enhances, but is not essential for, selfing and outcrossing in G. zeae, whereas no functional role was found for the other pair (ppg2/pre1).