The purpose of this study was to investigate possible laboratory contamination of Mycobacterium tuberculosis cultures which resulted in the misdiagnosis of tuberculosis. We have investigated three cases in which a patient's culture was positive for M. tuberculosis but there was not a high clinical suspicion for disease. In each instance, another patient with clinically obvious pulmonary tuberculosis had specimens cultured concurrently within the same clinical laboratory. The isolates from both the obvious cases of tuberculosis and the suspect cases were obtained through the State of Alabama TB Laboratory, but these isolates originated at a commercial laboratory, a community hospital laboratory, and at a university hospital. MTB isolates were fingerprinted by probing for the insertion sequence IS6110. With each of the three pairs of isolates (case and suspicious case), identical IS6110 banding patterns were found suggesting identical MTB strains. Because the patients were geographically separated, it is strongly suspected that laboratory contamination of M. tuberculosis cultures resulted in the three suspect cases being diagnosed with tuberculosis. These findings indicate that positive M. tuberculosis cultures resulting from laboratory contamination can occur.