Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of self-incompatibility. The S(11) and S(13) RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S(11) or S(13) pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S(11) RNase to match those in the S(13) RNase was sufficient to completely replace the S(11) phenotype with the S(13) phenotype. We now show that an S(11) RNase in which only three amino acid residues were modified to match those in the S(13) RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S(11) and S(13) pollen). Thus, S(12)S(14) plants expressing this hybrid S RNase rejected S(11), S(12), S(13), and S(14) pollen yet allowed S(15) pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S(13) allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dual-specific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one.