Simultaneous infection with two different strains of Mycobacterium tuberculosis has been demonstrated using phage typing. We report here the first case of mixed infection identified using IS6110-based genotyping of M. tuberculosis. The patient was diagnosed with pulmonary tuberculosis in February, 1991. The initial isolate of M. tuberculosis had two different genotype patterns (dark 7-band and light 14-band patterns). However, in a repeat isolate obtained several months later, only the 14-band pattern was visible. Exogenous reinfection and laboratory cross-contamination were unlikely because both genotype patterns were unique in the San Francisco database which includes over 1300 isolates of M. tuberculosis. This case demonstrates the importance of identifying mixed infections in the study of the molecular epidemiology of tuberculosis. Mixed infections could be confused with exogenous reinfection or laboratory cross-contamination, and important epidemiologic connections could be missed.