Cell wall deficient forms (CWD, spheroplasts) genetically indistinguishable from M. avium subsp. paratuberculosis have been isolated from patients with Crohn's Disease and Sarcoidosis. These CWD organisms may be important in the pathogenesis of these diseases, as well as Johne's disease in other animal species. However, CWD forms are extremely difficult to isolate and cultivate. When cultured in vitro, they generally revert to cell wall competent forms. Since sufficient quantities of CWD could not be obtained for experimentation, Naser and co- workers recently developed a method to chemically generate large numbers of M. avium subsp. paratuberculosis CWD. In this study, chemically generated M. avium subsp. paratuberculosis 19698 CWD were compared to cell wall competent M. avium subsp. paratuberculosis 19698 organisms. Organisms were evaluated by electron microscopy, chemotype profile (using matrix solid phase dispersion and thin-layer chromatography), silver stained SDS-PAGE gels, and Western blots. Marked differences in organism morphology, chemotype profile, presence of proteins and glycosylated compounds, and recognition of antigens by Johne's Disease positive and negative bovine control serum were detected between CWD and cell wall competent forms of M. avium subsp. paratuberculosis 19698.