Several investigators have recently described the isolation of slow growing mycobacteria from the tissues of patients with Crohn's disease (CD). The primary purpose of this study was to culture and identify mycobacteria from the intestines of patients with CD and other intestinal diseases (control tissues). The culture methods were designed to eliminate most rapid-growing mycobacteria and to enhance the isolation of slow growing mycobacteria. Eighty-two surgically resected intestinal tissue samples were cultured over a four-year period: 27 tissues were from CD patients and 55 from patients with other intestinal diseases. After 4-12 months of culture, five mycobacteria were isolated, but only two have been identified thus far. Both of these organisms appeared to have initially grown as spheroplasts, but revertant bacteria were cultivated after transfer into fresh media. Four of the mycobacteria were from CD tissues, and one isolate was from a control tissue. Two of the isolates have been identified as M. chelonei subsp. abscessus, strain 390 and M. paratuberculosis strain 410. This M. paratuberculosis is similar to the previously identified M. paratuberculosis strains isolated from other human intestinal tissues from patients with CD. Both strains 390 and 410 were inoculated into neonatal goats, but they failed to reproduce a CD-like disease. The isolation of four mycobacteria from 27 CD tissues and only one from 55 control tissues strengthens the findings of previous investigators and supports the hypothesis that mycobacteria may be etiologically associated with some cases of Crohn's disease.