The recognition specificity of the pollen ligand of self-incompatibility (SP11/SCR) was investigated using Brassica rapa transgenic plants expressing SP11 transgenes, and SP11 of Raphanus sativus S-21 was found to have the same recognition specificity as that of B. rapa S-9. In a set of three S haplotypes, whose sequence identities of SP11 and SRK are fairly high, R. sativus S-6 showed the same recognition specificity as Brassica oleracea S-18 and a slightly different specificity from B. rapa S-52. B. oleracea S-18, however, showed a different specificity from B. rapa S-52. Using these similar S haplotypes, chimeric SP11 proteins were produced by domain swapping. Bioassay using the chimeric SP11 proteins revealed that the incompatibility response induction activity was altered by the replacement of Region III and Region V. Pollen grains of Brassica transgenic plants expressing chimeric SP11 of the B. oleracea SP11-18 sequence with Region III and Region V from B. rapa SP11-52 (chimeric BoSP11-18[52]) were partially incompatible with the B. rapa S-52 stigmas, and those expressing the R. sativus SP11-6 sequence with Region III and Region V from B. rapa SP11-52 (chimeric RsSP11-6[52]) were completely incompatible with the stigmas having B. rapa S-52. However, the transgenic plant expressing chimeric RsSP11-6(52) also showed incompatibility with B. oleracea S-18 stigmas. These results suggest that Regions III and Region V of SP11 are important for determining the recognition specificity, but not the sole determinant. A possible process of the generation of a new S haplotype is herein discussed.