Drug-resistant Mycobacterium tuberculosis isolates are a major public health concern worldwide. There are few studies assessing tuberculosis (TB) resistance in Brazil. This study assessed the prevalence of resistance to the five first-line anti-TB drugs in TB isolates from HIV-infected patients in a tertiary care teaching hospital in southern Brazil. From September 1997 to July 2003, 398 TB complex isolates were included in the study. Resistance to one or more first-line anti-TB drugs was found in 71 (17.8%) of patients and was significantly more frequent among previously treated patients (12 [30.8%] of 39 patients) than new cases (59 [16.4%] of 359) (P=0.05). The highest resistance rates were found to isoniazid (9.9% overall; and 25.6% among previously treated patients). Multidrug-resistant TB was found in eight (2.0%) patients, with higher rates among previously treated patients than new cases: two (5.1%) patients vs. six (1.6%), respectively (P=0.18). Multidrug resistance and particularly isoniazid resistance rates among previously treated HIV patients are of great concern. Our findings indicate the need to reappraise regional TB treatment policies and support the recommendation for routine performance of in vitro TB susceptibility tests in all previously treated patients.