To assess the role of non-tuberculous mycobacteria (NTM) as a cause of tuberculosis-like diseases in Zambia, 167 chronically ill patients, hospitalized in three rural hospitals in Katete, Sesheke and Chilonga, were examined by microscopy and liquid culture for the presence of NTM. The percentages of patients with a positive culture for Mycobacterium tuberculosis complex were similar in the three geographical locations (19-25%). In contrast, the percentage of NTM ranged from 78% in Katete and 65% in Sesheke to 21% in Chilonga. Furthermore, the distribution of NTM species was different at the three geographical sites. In seven patients, true NTM-associated disease was suspected: five with Mycobacterium lentiflavum and two with Mycobacterium intracellulare. Analysis of possible risk factors indicated that the OR for NTM culture-positive sputum was significantly higher for patients living in Katete and Sesheke. Female gender and chest X-ray appearances of tuberculosis were independently associated with NTM culture-positive sputum. NTM colonization and disease in hospitalized, chronically ill patients in rural Zambia appear to be common.