Petunia inflata possesses S-RNase-based self-incompatibility (SI), which prevents inbreeding and promotes outcrossing. Two polymorphic genes at the S-locus, S-RNase and P. inflata S-locus F-box (Pi SLF), determine the pistil and pollen specificity, respectively. To understand how the interactions between Pi SLF and S-RNase result in SI responses, we identified four Pi SLF-like (Pi SLFL) genes and used them, along with two previously identified Pi SLFLs, for comparative studies with Pi SLF2. We examined the in vivo functions of three of these Pi SLFLs and found that none functions in SI. These three Pi SLFLs and two other Pi SLFs either failed to interact with S3-RNase (a non-self S-RNase for all of them) or interacted much more weakly than did Pi SLF2 in vitro. We divided Pi SLF2 into FD1 (for Functional Domain1), FD2, and FD3, each containing one of the Pi SLF-specific regions, and used truncated Pi SLF2, chimeric proteins between Pi SLF2 and one of the Pi SLFLs that did not interact with S3-RNase, and chimeric proteins between Pi SLF1 and Pi SLF2 to address the biochemical roles of these three domains. The results suggest that FD2, conserved among three allelic variants of Pi SLF, plays a major role in the strong interaction with S-RNase; additionally, FD1 and FD3 (each containing one of the two variable regions of Pi SLF) together negatively modulate this interaction, with a greater effect on interactions with self S-RNase than with non-self S-RNases. A model for how an allelic product of Pi SLF determines the fate of its self and non-self S-RNases in the pollen tube is presented.