The mating-type locus of , a representative edible mushroom, is highly complex because of allelic variations in the mating pheromone receptors (s) and the mating pheromones (s) in both the and subloci. The complexity of the mating-type locus, five subloci with five alleles of and nine and three subloci with 3 alleles of and five s, has led us to investigate the specificity of the PHB-RCB interaction because the interaction plays a key role in non-self-recognition. In this study, the specificities of PHBs to RCB1-2 and RCB1-4 from the sublocus and RCB2-1 from the sublocus were investigated using recombinant yeast strains generated by replacing , an endogenous yeast mating pheromone receptor, with the s. Fourteen synthetic PHBs with C-terminal carboxymethylation but without farnesylation were added to the recombinant yeast cells and the PHB-RCB interaction was monitored by the expression of the gene-a downstream gene of the yeast mating signal pathway. RCB1-2 () was activated by PHB1 (4.3-fold) and PHB2 (2.1-fold) from the sublocus and RCB1-4 () was activated by PHB5 (3.0-fold) and PHB6 (2.7-fold) from the sublocus and PHB13 (3.0-fold) from the sublocus. In particular, PHB3 from and PHB9 from showed strong activation of RCB2-1 of the sublocus by 59-fold. The RCB-PHB interactions were confirmed in the monokaryotic S1-10 strain of by showing increased expression of a downstream gene of the mating signal pathway and the occurrence of clamp connections after the treatment of PHBs. These results indicate that a single PHB can interact with a non-self RCB in a sublocus-specific manner for the activation of the mating pheromone signal pathways in .