Previous studies using DNA fingerprinting revealed three major groups of M. paratuberculosis. One of these groups, which in New Zealand is found principally in small ruminants and deer, is not readily isolated in primary culture on Herrold's egg yolk medium supplemented with mycobactin. In this study we evaluated the use of a liquid culture system (MGIT, Becton Dickinson) modified by the addition of mycobactin, for isolating M. paratuberculosis from lymph nodes of deer and sheep. A total of 85 frozen homogenates of tissues known to contain mycobacteria were cultured using MGITs and Herrold's egg yolk medium. The identity of isolates was confirmed as M. paratuberculosis using a PCR test based on the insertion element IS900. Further characterisation of isolates was carried out using a PCR test based on primers from IS900 which generated a PCR product with isolates from the "ovine" DNA group of M. paratuberculosis but not from the "bovine" DNA group. Growth of M. paratuberculosis was observed in the MGIT system after 3 to 37 days incubation in 17 of 18 samples containing large numbers of mycobacteria. In contrast, growth on Herrold's medium for these samples took a minimum of 21 days and only 3 of 18 had grown on the solid medium by 37 days. Examination of the isolates by PCR indicated that members of the "bovine" DNA group can be readily isolated from tissue samples using the MGIT system. Furthermore, some members of the "ovine" group can also be isolated from tissues using MGITs.