We have recently developed a nested PCR method which amplifies internal transcribed spacers (ITS) of the ribosomal RNA genes of Pneumocystis carinii. To determine whether this PCR method can be used to diagnose P. carinii infections, we examined 30 bronchoalveolar lavage (BAL) specimens that were shown microscopically to contain P. carinii organisms by the P. carinii ITS PCR (Pc-ITS-PCR) and five other PCR methods that have been described for detecting P. carinii in clinical specimens. The targets of these PCR methods are portions of 18S rRNA, mitochondrial (mt) rRNA, 5S rRNA, thymidylate synthase (TS), and dihydrofolate reductase (DHFR). We also examined five different fungi, including Saccharomyces cerevisiae, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus to determine the specificity of these six PCR methods for P. carinii. All 30 BAL specimens were positive by both the Pc-ITS-PCR and the 18S rRNA gene PCR, whereas only 26 (87%), 18 (60%), 10 (33%), and 7 (23%) of 30 BAL specimens were positive by mt rRNA gene PCR, TS gene PCR, 5S rRNA gene PCR, and DHFR gene PCR, respectively. Although the 18S rRNA gene PCR had the same sensitivity as the Pc-ITS-PCR, it nonspecifically amplified S. cerevisiae and C. albicans. The TS gene PCR also produced false-positive PCR results with C. albicans and C. neoformans. None of the other PCR methods (Pc-ITS-PCR, mt rRNA gene, 5S rRNA gene, and DHFR gene PCR) amplified the control fungal DNA. Considering both sensitivity and specificity, we conclude that Pc-ITS-PCR is the most effective of the six PCR methods evaluated in this study for the detection of P. carinii in BAL specimens.