False-positive Mycobacterium tuberculosis cultures are a benchmark for the quality of laboratory processes and patient care. We studied the incidence of false-positive cultures, risk factors, and consequences for patients during the period from 1993 to 2000 in 44 peripheral laboratories in The Netherlands. The national reference laboratory tested 8,889 M. tuberculosis isolates submitted by these laboratories. By definition, a culture was false positive (i) if the DNA fingerprint of the isolate was identical to that of an isolate from another patient processed within 7 days in the same laboratory, (ii) if the isolate was taken from a patient without clinical signs of tuberculosis, and/or (iii) if the false-positive test result was confirmed by the peripheral laboratory and/or the public health tuberculosis officer. We identified 213 false-positive cultures (2.4%). The overall incidence of false-positive cultures decreased over the years, from 3.9% in 1993 to 1.1% in 2000. Laboratories with false-positive cultures more often processed less than 3,000 samples per year (P < 0.05). Among 110 patients for whom a false-positive culture was identified from 1995 to 1999, we found that for 36% of the patients an official tuberculosis notification had been provided to the appropriate public health services, 31% of the patients were treated, 14% of the patients were hospitalized, and a contact investigation had been initiated for 16% of the patients. The application of DNA fingerprinting to identify false-positive M. tuberculosis cultures and the provision of feedback to peripheral laboratories are useful instruments to improve the quality of laboratory processes and the quality of medical care.