All New York City patients whose cultures yielded Mycobacterium tuberculosis with isolated resistance to rifampin in 1993 and 1994 were included in this study. Of the 96 patients, 48 (50%) had primary resistance, 32 (33%) had acquired resistance, and 16 (17%) had unclassified resistance; 66% had histories of illicit drug use, and 79% were infected with human immunodeficiency virus (HIV). The median time to emergence of resistance was 40 weeks among the 32 patients with acquired resistance. Each of the HIV-infected patients with acquired resistance (cases, n = 29) was matched to two HIV-infected patients who had disease due to fully susceptible M. tuberculosis (controls, n = 58). In multivariate analysis, factors associated with the emergence of rifampin resistance were as follows: a sputum smear positive for acid-fast bacilli, advanced immunosuppression, and nonadherence to therapy.