S-RNase-based self-incompatibility has been identified in three flowering plant families, including the Solanaceae, and this self/non-self recognition mechanism between pollen and pistil is controlled by two polymorphic genes at the S-locus, S-RNase and S-locus F-box (SLF). S-RNase is produced in the pistil and taken up by pollen tubes in a non-S-haplotype-specific manner. How an allelic product of SLF interacts with self and non-self S-RNases to result in growth inhibition of self pollen tubes is not completely understood. One model predicts that SLF targets non-self S-RNases for ubiquitin/26S proteasome-mediated degradation, thereby only allowing self S-RNase to exert cytotoxic activity inside a pollen tube. To test this model, we studied whether any of the 20 lysine residues in S3-RNase of Petunia inflata might be targets for ubiquitination. We identified six lysines near the C-terminus for which mutation to arginine significantly reduced ubiquitination and degradation of the mutant S3-RNase, GST:S3-RNase (K141–164R) in pollen tube extracts. We further showed that GST:S3-RNase (K141–164R) and GST:S3-RNase had similar RNase activity, suggesting that their degradation was probably not caused by an ER-associated protein degradation pathway that removes mis-folded proteins. Finally, we showed that PiSBP1 (P. inflata S-RNase binding protein 1), a potential RING-HC subunit of the PiSLF (P. inflata SLF)-containing E3-like complex, could target S-RNase for ubiquitination in vitro. All these results suggest that ubiquitin/26S proteasome-dependent degradation of S-RNase may be an integral part of the S-RNase-based self-incompatibility mechanism.