Pneumocystis jiroveci is an important agent of pneumonia in immunocompromised hosts. Usually, this pathogen is detected by Giemsa or direct fluorescence stains of bronchoalveolar lavage (BAL) fluids. Microscopic methods, however, have 2 disadvantages. P. jiroveci is not stable outside the human body, which means that slow sample transport might result in false-negative results. Additionally, exact quantification, which is needed for therapy monitoring, is not possible. In this study, we developed a real-time polymerase chain reaction assay for the LightCycler. Two Pneumocystis-specific TaqMan systems, one based on the sequence of the 5.8S ribosomal gene and another one targeting the dihydrofolate reductase gene were evaluated. Additionally, the amount of human DNA in the sample was measured by a TaqMan assay based on the human albumin gene, allowing assessment of sample quality and quantification normalized on sample concentration. For clinical evaluation, 69 BAL specimens from 26 positive patients as well as 60 negative controls were tested. Both systems were able to detect all proven cases of Pneumocystis pneumonia. Differentiation of carriage, asymptomatic reactivation, and clinical infection as well as normalized quantification by calculating the ratio of Pneumocystis DNA to human DNA are discussed.