We investigated the utility of PCR to detect Burkholderia cepacia directly in sputum samples at two cystic fibrosis (CF) centers serving children and adults. Following liquefaction of the sputa by using N-acetyl-l-cysteine, DNA was isolated and analyzed by PCRs with three different primer pairs directed toward bacterial rRNA loci. Two primer pairs were putatively specific for B. cepacia. The other pair, which universally amplifies a band from all bacteria, served as a control. Sputum samples were obtained from 219 patients and analyzed independently by culture and by PCR to detect B. cepacia. The analyses were performed blinded with respect to each other. The results of the PCR with sputa demonstrated that the primers directed to the 16S loci demonstrated approximately 95% concordance with culture results and were more specific than those amplifying the 16S to 23S spacer region. In addition, the 16S primer pair putatively identified B. cepacia in seven patients whose sputa were culture negative at this time. Of these culture-negative patients, five had sputum samples that were culture positive for B. cepacia either prior or subsequent to this study. The results of this study indicate the utility of PCR as a diagnostic method for the rapid identification of B. cepacia in sputum samples of CF patients. We anticipate that improvements in our taxonomic understanding may allow the design of more specific primers for detection of each species of the B. cepacia complex in sputum samples.