OBJECTIVE To investigate possible cross-contamination of laboratory specimens, as suggested by an increased incidence of newly diagnosed patients with tuberculosis, many of whom had all negative smears for acid-fast bacilli and only one positive Mycobacterium tuberculosis culture referred to as "negative smears, one positive" or NSOP. METHODS Medical-record reviews were performed for all patients with NSOP results diagnosed at this facility within a 9-month period. Laboratory logbooks were reviewed for all isolates processed; DNA fingerprinting was performed on available isolates. RESULTS Of 80 patients with NSOP results, 45 (56%) were found to have false-positive cultures resulting from laboratory contamination with H37Ra, an avirulent stock strain of Mycobacterium tuberculosis. CONCLUSIONS Laboratory cross-contamination resulted in the false diagnosis of tuberculosis in at least 45 individuals. Use of the Mycobacteria Growth Indicator Tube may have contributed to these contamination incidents by detecting small numbers of contaminating mycobacteria that may not have been detected with less sensitive media.