Background Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrP^res. Results Optimal PrP^res detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrP^res concentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype–VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171–allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002–2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrP^res, but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrP^res in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrP^res bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrP^res, BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrP^res was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study. Conclusion The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products.