Mycobacterium avium subsp. paratuberculosis is distinguished from other mycobacterial species by its dependence on the iron-chelator factor mycobactin and its prolonged incubation period when grown in vitro. Traditionally, a very low rate of isolation has been considered characteristic of sheep strains of this mycobacterium species. In the present study, a comparison was made of the performance of two media, Löwenstein-Jensen and Middlebrook 7H11/OADC both with and without mycobactin, on the isolation of M. a. paratuberculosis from ovine cases of the disease. A high isolation rate in both media was observed. Moreover, our results indicated that mycobactin dependence of sheep strains of M. a. paratuberculosis is medium related, being dependent on Löwenstein-Jensen and independent on Middlebrook 7H11/OADC.