Interactions between Arabidopsis thaliana and the downy mildew fungus Peronospora parasitica provide a model system to study the genetic and molecular basis of plant-pathogen recognition. With the use of the Noco2 isolate of P. parasitica, the reaction phenotypes of 46 accessions of Arabidopsis were examined and 31 accessions exhibited resistance. Resistance phenotypes examined ranged from distinct necrotic pits or flecks to a weak necrosis accompanied by late and sparse fungal sporulation. Segregating populations generated from crosses between the susceptible accession Col-0 and the resistant accessions Ws-0, Pr-0, Oy-0, Po-1, Bch-1, Ge-1, Di-1, Ji-1, and Te-0 were also screened with Noco2. The genetic data were consistent with the presence of single resistance (RPP) loci in all of these accessions except Oy-0, in which resistance was inherited as a digenic trait. As a first step to molecular cloning, the map positions of four resistance loci were determined. These have been designated RPP14.1 from Ws-0, RPP14.2 from Pr-O, and RPP14.3 and RPP5.2 from Oy-0. RPP14.1 was mapped to a 3.2-cM interval on chromosome 3 that is linked to a region between the markers Gl-1 and m249 known to contain other P. parasitica resistance specificities. RPP14.2 from Pr-0 and RPP14.3 from Oy-0 were also positioned in this interval. Moreover, RPP14.1 and RPP14.2 showed linkage of < 0.05 cM, suggesting possible allelism. The second RPP locus from Oy-0, RPP5.2, was located on chromosome 4 and exhibited strong linkage (< 2 cM) to RRP5.1, a locus previously identified in the Arabidopsis accession Landsberg-erecta. The results reinforce evidence for RPP gene clustering in the Arabidopsis genome and provide new targets for cloning and examination of RPP gene structure, function, allelic variation, and organization within defined loci.