We have used map-based approaches to clone a locus containing two genes, Avr1b-1 and Avr1b-2, required for avirulence of the oomycete pathogen Phytophthora sojae (Kaufmann & Gerdemann) on soybean plants carrying resistance gene Rps1b. Avr1b-1 was localized to a single 60-kb bacterial artificial chromosome (BAC) clone by fine-structure genetic mapping. Avr1b-1 was localized within the 60-kb region by identification of an mRNA that is expressed in a race-specific and infection-specific manner and that encodes a small secreted protein. When the Avr1b-1 protein was synthesized in the yeast Pichia pastoris and the secreted protein infiltrated into soybean leaves, it triggered a hypersensitive response specifically in host plants carrying the Rps1b resistance gene. This response eventually spread to the entire inoculated plant. In some isolates of P. sojae virulent on Rps1b-containing cultivars, such as P7081 (race 25) and P7076 (race 19), the Avr1b-1 gene had numerous substitution mutations indicative of strong divergent selection. In other isolates, such as P6497 (race 2) and P9073 (race 25), there were no substitutions in Avr1b-1, but Avr1b-1 mRNA did not accumulate. Genetic complementation experiments with P6497 revealed the presence of a second gene, Avr1b-2, required for the accumulation of Avr1b-1 mRNA. Avr1b-2 was genetically mapped to the same BAC contig as Avr1b-1, using a cross between P7064 (race 7) and P6497. The Avr1k gene, required for avirulence on soybean cultivars containing Rps1k, was mapped to the same interval as Avr1b-1.