Historically, infections caused by Mycobacterium tuberculosis have been treated simultaneously with isoniazid and rifampin. As a consequence of this combined therapy, strains resistant only to rifampin were rarely recovered. However, recently there has been an increasing number of reports describing HIV-positive patients infected with mono-rifampin-resistant M. tuberculosis strains. Organisms cultured from seven patients (including six with AIDS) with infections caused by mono-rifampin-resistant M. tuberculosis, and seen at one New York City hospital, were analyzed by molecular techniques to test the hypothesis that dissemination of a single clone had occurred. IS6110 DNA fingerprinting and automated DNA sequencing of a region of the RNA polymerase beta subunit structural gene (rpoB) containing mutations that confer rifampin resistance showed that all organisms independently acquired the mono-rifampin-resistant phenotype. Molecular analysis of mono-rifampin-resistant organisms cultured from 13 additional patients in New York City confirmed independent strain origin. The data rule out the possibility of person-to-person strain transmission among these patients, and they suggest that host factors such as poor compliance with antituberculosis medications or decreased absorption of rifampin have been a driving force in the origin of these strains.