SETTING: Tygerberg Children's Hospital, Cape Town, South Africa. OBJECTIVE: To determine the prevalence and trend of drug resistance and human immunodeficiency virus (HIV) co-infection among children with culture-confirmed tuberculosis (TB). METHOD: Prospective surveillance from March 2007 to February 2009, compared to three previous surveys (1994-1998, 2003-2005, 2005-2007). Drug susceptibility testing (DST) against isoniazid (INH) and rifampicin (RMP) was performed using genotypic and phenotypic testing. If multidrug-resistant TB (MDR-TB) was detected, further DST against ethambutol (EMB) and second-line drugs was performed. RESULTS: A total of 294 children with a median age of 26 months (range 3 days-13 years) were diagnosed with culture-confirmed TB. DST results were available for 292 (99.3%); 41 (14%) were INH-resistant, including 26 (8.9%) with MDR-TB. Four children (1.4%) had RMP monoresistance. EMB resistance was present in 12/24 (50%) MDR-TB cases tested. Two isolates were resistant to ofloxacin; none had extensively drug-resistant TB. Of those tested, 29% (63/217) were HIV-infected. Any resistance to RMP increased between 1994 and 2009 (P < 0.001), as did RMP monoresistance (P = 0.009) and MDR-TB (P < 0.001). Sensitivity was 87.5% and specificity 100% for genotypic compared to phenotypic testing for INH resistance. CONCLUSIONS: RMP, and consequently multidrug, resistance is increasing among children with TB in this setting. EMB resistance is common among children with resistance to RMP and INH.