Pneumocystis carinii pneumonia (PCP) remains a major cause of morbidity and mortality in immunocompromised patients, including those infected with human immunodeficiency virus (HIV). The advent of real-time PCR technology offers the potential for rapid PCR results for the detection of P. carinii. In this report we describe the modification and evaluation of an existing PCR-based method for the detection of P. carinii DNA, into a real-time PCR assay suitable for use with the LightCycler system. Twenty eight induced sputum and bronchial washing specimens from 28 patients were tested by both a conventional PCR assay and a real-time PCR assay. Twelve specimens (42.9%) were positive in both the conventional and real-time PCR assays and sixteen (57.1%) were negative in both assays. The melting points of the amplified P. carinii DNA product obtained by melting curve analysis by the LightCycler of all P. carinii positive specimens ranged from 81.5 degrees C to 83.9 degrees C. There were no discordant results between the two assays for any of the specimens tested and results were available within 2 h for the real-time PCR assay compared to up to 11 h for the conventional PCR assay.