To evaluate the clinical use of a polymerase chain reaction (PCR) assay for diagnosis of Pneumocystis carinii pneumonia (PCP) using samples collected non-invasively, the Internal Transcribed Spacers (ITSs) nested PCR was performed on 148 samples from 40 subjects. Bronchoalveolar lavage (BAL) fluid sera, gargled oropharyngeal washes, and peripheral blood mononuclear cells (PBMC) from 14 AIDS patients (mean age, 35.6 years; mean CD4+ cell count, 49.2 cells/mm3) with proven PCP and from 13 HIV-seropositive controls (mean age, 34.6 years; mean CD4+ cell count, 107.3 cells/mm3) with other AIDS-related opportunistic infections were evaluated. Sera and oropharyngeal samples were also collected from 13 HIV-seronegative health care personnel working in an infectious disease ward for use as negative controls. The ITSs nested PCR confirmed the morphological diagnosis of PCP in all patients when BAL fluid was tested (100% sensitivity). This technique also detected Pneumocystis carinii DNA in oropharyngeal samples from 78.6% of patients, in sera from 71.4% of patients, in PBMC from 35.7% of patients. When all results obtained after ITSs nested PCR were considered together for the same patient, the sensitivity for PCP diagnosis was 100% for blood and oropharyngeal samples (gargled saline), as confirmed by subsequent BAL. All samples collected noninvasively from 26 of 26 controls were negative using ITSs nested PCR (100% specificity).