A polymerase chain reaction (PCR) that amplifies the internal transcribed spacers of rRNA genes of Pneumocystis carinii (Pc-ITS-PCR) was compared with dihydrofolate reductase (DHFR) gene PCR to determine its usefulness in the diagnosis of Pneumocystis carinii pneumonia. All specimens from non-P. carinii pneumonia AIDS patients were both Pc-ITS-PCR- and DHFR PCR-negative. Twenty-six (96%) of 27 bronchoalveolar lavage specimens from P. carinii pneumonia patients were Pc-ITS-PCR-positive, and all 27 of these patients had at least 1 serum sample positive by Pc-ITS-PCR. In contrast, DHFR PCR detected P. carinii in only 4 (17%) of 24 P. carinii-positive lavage specimens and 2 (10%) of the 20 sera from P. carinii pneumonia patients. These results suggest that serum Pc-ITS-PCR may be useful for the diagnosis of P. carinii pneumonia.