The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC-and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.T HE switch from an outcrossing mode of mating to selffertility was a major transition in the evolutionary history of Arabidopsis thaliana. Recent studies have shown that this switch was accompanied by multiple independent losses of self-incompatibility (SI), the major mechanism that promotes outcrossing in the Brassicaceae Shimizu et al. 2008; Boggs et al. 2009a). In this family, SI is controlled by numerous haplotypes of the S locus. Within each S-locus haplotype (hereafter S haplotype), are two genes that determine specificity in the SI response: one gene encodes the stigma-expressed S-locus receptor kinase (SRK) and the other encodes the pollen coat-localized ligand for SRK, the S-locus cysteine-rich (SCR) protein. The SRK and SCR proteins are highly polymorphic and co-evolving proteins (Sato et al. 2002) and their haplotype-specific interaction is responsible for the specific recognition and inhibition by the stigma epidermis of self-related pollen (i.e., pollen derived from the same flower, other flowers on the same plant, or plants expressing the same S haplotype) (reviewed in Rea and Nasrallah 2008). Consequently, an understanding of the genetic events associated with the switch to self-fertility in the A. thaliana lineage was sought through analysis of SRK and SCR sequences harbored by various A. thaliana geographical accessions (Kusaba et al. 2001;Shimizu et al. 2004Shimizu et al....