Molecular methods have the potential to be a valuable tool in the diagnosis of Johne's disease (JD). However, the direct application of IS900 PCR to faeces is hindered by the presence of constituents in the faecal material inhibitory to the Taq DNA polymerase enzyme. An immunomagnetic PCR (IMS-PCR) technique, recently developed at Queen's University to facilitate the selective isolation of M. paratuberculosis from milk, was adapted for application to faeces. Faeces samples were diluted (1:5) in PBS-0.05% Tween 20, shaken vigorously by hand and then centrifuged (300 x g for 3 min) to quickly sediment particulate matter. One ml of the resultant supernatant was subjected to immunomagnetic separation (IMS). Positive PCR signals were obtained after IMS from faeces spiked with as few as 10 CFU of M. paratuberculosis per g. In contrast, when IMS-PCR was applied to stomached faeces samples, a PCR signal was only obtained when high numbers of M. paratuberculosis were present, indicating that low-speed centrifugation more effectively removed particulate matter. A comparative blind trial was carried out on 95 faeces samples, chiefly of bovine origin. Each faeces sample was subjected to microscopic examination after Ziehl-Neelsen (ZN) staining, IMS-PCR, and HPC decontamination and culture. Results showed that IMS-PCR detected more infected samples than ZN smear, and as many infected samples as culture. This IMS-PCR assay could aid the more rapid diagnosis of JD.