S proteins, pistil-specific ribonucleases that cosegregate with S alleles, have previously been shown to control rejection of self-pollen in Petunia inflata and Nicotiana alata, two solanaceous species that display gametophytic self-incompatibility. The ribonuclease activity of S proteins was thought to degrade RNA of self-pollen tubes, resulting in the arrest of their growth in the style. However, to date no direct evidence has been obtained. Here, the ribonuclease activity of S3 protein of P. inflata was abolished, and the effect on the pistil's ability to reject S3 pollen was examined. The S3 gene was mutagenized by replacing the codon for His-93, which has been implicated in ribonuclease activity, with a codon for asparagine, and the mutant S3 gene was introduced into P. inflata plants of S1S2 genotype. Two transgenic plants produced a level of mutant S3 protein comparable to that of the S3 protein produced in self-incompatible S1S3 and S2S3 plants, yet they failed to reject S3 pollen. The mutant S3 protein produced in these two transgenic plants did not exhibit any detectable ribonuclease activity. We have previously shown that transgenic plants (S1S2 plants transformed with the wild-type S3 gene) producing a normal level of wild-type S3 protein acquired the ability to reject S3 pollen completely. Thus, the results reported here provide direct evidence that the biochemical mechanism of gametophytic self-incompatibility in P. inflata involves the ribonuclease activity of S proteins.