A striking increase in the numbers of cultures positive for Mycobacterium tuberculosis was noticed in a mycobacterial reference laboratory in Campinas, Sao Paulo State, Brazil, in May 1995. A contaminated bronchoscope was the suspected cause of the increase. All 91 M. tuberculosis isolates grown from samples from patients between 8 May and 18 July 1995 were characterized by spoligotyping and IS6110 fingerprinting. Sixty-one of the 91 isolates had identical spoligotype patterns, and the pattern was arbitrarily designated S36. The 61 specimens containing these isolates had been processed and cultured in a 21-day period ending on 1 June 1995, but only 1 sample was smear positive for acid-fast bacilli. The patient from whom this sample was obtained was considered to be the index case patient and had a 4+ smear-positive lymph node aspirate that had been sent to the laboratory on 10 May. Virtually all organisms with spoligotype S36 had the same IS6110 fingerprint pattern. Extensive review of the patients' charts and investigation of laboratory procedures revealed that cross-contamination of specimens had occurred. Because the same strain was grown from all types of specimens, the bronchoscope was ruled out as the outbreak source. The most likely source of contamination was a multiple-use reagent used for specimen processing. The organism was cultured from two of the solutions 3 weeks after mock contamination. This investigation strongly supports the idea that M. tuberculosis grown from smear-negative specimens should be analyzed by rapid and reliable strain differentiation techniques, such as spoligotyping, to help rule out laboratory contamination.