We reviewed medical records of patients without known HIV and with positive cultures for nontuberculous mycobacteria (NTM) isolated during 2000-2003 from 1 large hospital in New York, New York. Overall, 505 patients had positive NTM cultures; 119 (24%) met the criteria for NTM disease. The difference between demographic characteristics of case-patients in our study (66% female, 61% white, and 59% > 60 years of age) and those of the base population as determined by regional census data was statistically significant. Estimated incidences for positive cultures, all disease, and respiratory tract disease were 17.7, 2.7, and 2.0 per 100,000 persons, respectively. More patients with rapidly growing mycobacteria (61%), Mycobacterium kansasii (70%), or M. marinum (100%) met criteria for disease than did patients with M. avium complex (MAC) (27%, (p < 0.01). NTM disease in patients without HIV is increasing. Laboratory-based surveillance may be useful for detecting non-MAC and non-respiratory tract disease.