A novel heminested IS6110 polymerase chain reaction (PCR) assay was evaluated as a tool for diagnosing tuberculosis in 222 children. In an analysis of 392 specimens (gastric aspirates, nasopharyngeal aspirates, and sputum samples), results of PCR were compared with those of 3 culture methods, acid-fast bacillus (AFB) staining, and clinical assessment by the Stegen-Toledo score. The sensitivity of PCR (67%) was comparable to that of the 3-culture method (71%) and was significantly higher than that of Löwenstein-Jensen culture (54%) or AFB stain (42%) for children with highly probable tuberculosis. PCR detection rates for culture-positive specimens were 100% for smear-positive samples and 76.7% for smear-negative samples. The specificity of PCR was 100% in control children. Compared with culture, PCR demonstrated a sensitivity of 90.4%, a positive predictive value of 89%, a specificity of 94%, and a negative predictive value of 95% (κ = .85). With clinical assessment as the standard, PCR had a sensitivity of 71%, a positive predictive value of 92%, a specificity of 95%, and a negative predictive value of 79% (κ = .67). PCR is a rapid and sensitive method for the early diagnosis of pediatric tuberculosis.