Sexual reproduction in many flowering plants involves self-incompatibility (SI), which is one of the most important systems to prevent inbreeding. In many species, the self-/nonself-recognition of SI is controlled by a single polymorphic locus, the S-locus. Molecular dissection of the S-locus revealed that SI represents not one system, but a collection of divergent mechanisms. Here, we discuss recent advances in the understanding of three distinct SI mechanisms, each controlled by two separate determinant genes at the S-locus. In the Brassicaceae, the determinant genes encode a pollen ligand and its stigmatic receptor kinase; their interaction induces incompatible signaling(s) within the stigma papilla cells. In the Solanaceae-type SI, the determinants are a ribonuclease and an F-box protein, suggesting the involvement of RNA and protein degradation in the system. In the Papaveraceae, the only identified female determinant induces a Ca2+-dependent signaling network that ultimately results in the death of incompatible pollen.