Background: The EphB2 gene was recently implicated as a prostate cancer (PC) tumour suppressor gene, with somatic inactivating mutations occurring in ,10% of sporadic tumours. We evaluated the contribution of EphB2 to inherited PC susceptibility in African Americans (AA) by screening the gene for germline polymorphisms. Methods: Direct sequencing of the coding region of EphB2 was performed on 72 probands from the African American Hereditary Prostate Cancer Study (AAHPC). A case-control association analysis was then carried out using the AAHPC probands and an additional 183 cases of sporadic PC compared with 329 healthy AA male controls. In addition, we performed an ancestry adjusted association study where we adjusted for individual ancestry among all subjects, in order to rule out a spurious association due to population stratification. Results: Ten coding sequence variants were identified, including the K1019X (3055ART) nonsense mutation which was present in 15.3% of the AAHPC probands but only 1.7% of 231 European American (EA) control samples. We observed that the 3055ART mutation significantly increased risk for prostate cancer over twofold (Fisher's two sided test, p = 0.003). The T allele was significantly more common among AAHPC probands (15.3%) than among healthy AA male controls (5.2%) (odds ratio 3.31; 95% confidence interval 1.5 to 7.4; p = 0.008). The ancestry adjusted analyses confirmed the association. Conclusions: Our data show that the K1019X mutation in the EphB2 gene differs in frequency between AA and EA, is associated with increased risk for PC in AA men with a positive family history, and may be an important genetic risk factor for prostate cancer in AA.