In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S2 and S6. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S6 genotype and 350 kb in the S2 genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.