Self-incompatibility in the Brassicaceae is controlled by multiple haplotypes encoding the pollen ligand (S-locus protein 11, SP11, also known as S-locus cysteine-rich protein, SCR) and its stigmatic receptor (S-receptor kinase, SRK). A haplotype-specific interaction between SP11/SCR and SRK triggers the self-incompatibility response that leads to self-pollen rejection, but the signalling pathway remains largely unknown. Here we show that Ca(2+) influx into stigma papilla cells mediates self-incompatibility signalling. Using self-incompatible Arabidopsis thaliana expressing SP11/SCR and SRK, we found that self-pollination specifically induced an increase in cytoplasmic Ca(2+) ([Ca(2+)]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca(2+) increase, which was inhibited by D-(-)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca(2+)]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca(2+) increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca(2+) influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.