An immunomagnetic separation (IMS) technique was developed to facilitate the selective recovery of Mycobacterium paratuberculosis from milk. Purified rabbit polyclonal antibody (IgG) raised against radiation-killed intact M. paratuberculosis cells was used to coat Sheep anti-rabbit IgG Dynabeads®. Trials showed that IMS selectively recovered M. paratuberculosis from milk spiked with 10-100 CFU of M. paratuberculosis when 10 µl of coated beads (10⁶ beads) were added to 1 ml of milk and incubated for 30 min at RT. During IMS components of the milk inhibitory to the PCR reaction are effectively removed and therefore IS900 PCR can be successfully applied to milk. Template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100°C for 15 min. Centrifugation (2,500 x g for 20 min) was employed to concentrate larger volumes of milk (10 and 50 ml) prior to IMS in order to increase the sensitivity of the IMS-PCR assay. IMS-PCR was capable of detecting 10³ CFU of M. paratuberculosis per 50 ml of milk (equivalent to 20 CFU/ml). A blind trial (40 milk samples), consisting of spiked and unspiked, raw and laboratory pasteurised milk samples, showed that IMS-PCR correctly identified spiked milk samples before and after laboratory-pasteurisation. Further studies confirmed that IMS-PCR was able to detect natural M. paratuberculosis infection in a raw goat milk sample and several raw sheep milk samples from a herd with a history of Johne's disease. The development and evaluation of this novel IMS-PCR technique will be described.