The aim of the study was to assess the utility of the polymerase chain reaction (PCR) assay in blood and urine for the diagnosis of tuberculosis (TB). We prospectively evaluated the usefulness of PCR performed in blood and urine samples from patients with proved or probable TB compared with a control group of patients. The PCR technique was performed using IS6110 primers. We included in the study 57 patients (43 with definite TB and 14 with probable TB) and 26 controls. Blood and urine samples were drawn at the time of microbiologic diagnosis and 3, 6, 9, and 12 months later. Cultures were positive in the early period (<1 month after treatment) in 11 of 57 patients (19%) with probable or definite TB, in comparison with 42% of patients (24/57) who yielded a positive PCR (P = 0.02). Urine samples increased the sensitivity of PCR determination in blood samples by 10%. The PCR in blood and/or urine was positive in 41% of patients with pulmonary TB, in 36% of patients with extrapulmonary TB, and in 50% of patients with disseminated TB. Mycobacterium tuberculosis was still detectable by PCR in 5 of 13 patients with cured TB after 1 or more months of antituberculous treatment. The PCR detection of M. tuberculosis in blood and urine samples is useful for the diagnosis of different clinical forms of TB, mostly in those patients in which sample extraction is difficult or requires aggressive techniques. The sensitivity of this technique could be improved studying more than 1 sample in each patient, even after initiating an antituberculous treatment.