Background: The frequency of clinical isolation of nontuberculous mycobacteria (NTM) in the Netherlands is increasing, but its clinical relevance is often uncertain. We assessed the frequency and clinical relevance of isolation of NTM in four associated hospitals in a single region in the Netherlands. Methods: We retrospectively reviewed medical files of all patients from whom NTM were isolated between January 1999 and January 2005. We used diagnostic criteria for nontuberculous mycobacterial disease published by the American Thoracic Society (ATS) to determine clinical relevance. Results: We found 232 patients from whom in a 91% majority, NTM were isolated from the respiratory tract. Patients were mostly white men, with an average age of 60 years and pre-existing pulmonary disease. Twenty-five percent (n=53) of 212 patients with pulmonary isolates met the ATS diagnostic criteria for pulmonary NTM disease; this percentage differed by species. Most patients were treated with rifampicin, ethambutol and clarithromycin. Treatment outcome for pulmonary NTM disease was suboptimal but differed by species; whereas overall 67% of treated patient improved, this was only 50% for those with pulmonary Mycobacterium avium disease. Lymphadenitis was the most common extrapulmonary disease type. Conclusions: Twenty-five percent of all patients with pulmonary NTM isolates met the ATS criteria. Clinical relevance differs by species. NTM isolation increases over time. Species distribution differs from neighboring countries and the M. avium complex isolates have traits different from those reported in the United States. Adherence to diagnostic and treatment guidelines can be improved.