The self-incompatibility ( S) loci from the Solanaceae, Rosaceae and Scrophulariaceae encode a class of ribonucleases, known as S RNases, which have been shown to control the pistil expression of self-incompatible reaction. In the former two families, the S loci have been shown to be located near centromere. However, the chromosomal location of the S locus in Antirrhinum, a species of the Scrophulariaceae, is not known. To determine its chromosomal location and genomic organization, an S-2 RNase gene and its corresponding 63 kb BAC clone were separately used for fluorescence in situ hybridization (FISH) of mitotic metaphase chromosomes of a self-incompatible Antirrhinum line Of S2S5. The results showed that the S-2 RNase detected a doublet signal near the centromere of the smallest chromosome (2n = 16). Two separate doublet signals of the tested BAC sequence were shown on both sides of the centromeres of all eight pairs of the chromosomes, suggesting that the Antirrhinum S locus is located in a pericentromeric region. Furthermore, a retrotransposon, named RIS1 (retrotransposon in the S locus), which has not been identified yet in. Antirrhinum, was found next to S-2 RNase. Taken together, the centromeric location of the S locus from the three S-RNase-based self-incompatible families provides a further support on a common origin of their evolution as well as suppressed recombination.