We identified two S-allele-associated proteins (S-proteins) in a self-compatible cultivar of Petunia hybrida based on their segregation in F1 hybrids between P. hybrida and its self-incompatible relative, Petunia inflata (with S2S2 genotype), and in selfed progeny of P. hybrida. These two S-proteins, designated Sx-protein (24 kDa) and So-protein (31 kDa), are pistil specific, and their expression follows a temporal and spatial pattern similar to that of S-proteins characterized in self-incompatible solanaceous species. Their amino-terminal sequences also share a high degree of similarity with those of solanaceous S-proteins. Selfing of P. hybrida yielded plants with SoSo,SxSo, and SxSx genotypes in an approximately 1:2:1 ratio, indicating that the Sx-and So-alleles, though expressed in the pistil, failed to elicit a self-incompatibility response. The S2-allele of P. inflata is expressed in all the F1 hybrids, rendering them capable of rejecting pollen bearing the S2-allele. The So-allele is not functional in the F1 hybrids, because all the F1 progeny with S2So genotype are self-compatible. However, in F1 hybrids with S2Sx genotype, approximately half are self-incompatible and half are self-compatible, indicating that the function of the Sx-allele depends on the genetic background. These results strongly suggest that the presence of functional S-alleles alone is not sufficient for expression of a self-incompatibility phenotype, and reaffirm the multigenic nature of gametophytic self-incompatibility suggested by earlier genetic studies.