Flowering plants have evolved various stratagems to prevent inbreeding and promote outcrosses. One such mechanism, gametophytic self-incompatibility, provides a genetic barrier to self-fertilization, and in the simplest cases is controlled by the highly polymorphic S locus. Growth of a pollen tube in the style is arrested when the S allele carried by the pollen matches one of the two S alleles carried by the pistil. Putative S allele proteins of the pistil have been identified in several solanaceous species based on their co-segregation with S alleles, and they have been shown to be ribonucleases. So far, there has been only correlative or indirect evidence for the claim that these S allele-associated proteins (S proteins) are involved in recognition and rejection of self pollen. Here we show that inhibition of synthesis of S3 and S2 proteins in Petunia inflata plants of S2S3 genotype by the antisense S3 gene resulted in failure of the transgenic plants to reject S3 and S2 pollen. We further show that expression of the transgene encoding S3 protein in P. inflata plants of S1S2 genotype confers on the transgenic plants the ability to reject S3 pollen. The self-incompatibility behaviour of the pollen was not affected by the transgene in either set of experiments. Taken together, these findings provide direct in vivo evidence that S proteins control the self-incompatibility behaviour of the pistil.