Pneumocystis jirovecii pneumonia (PCP) is an acute and life-threatening lung disease caused by the fungus Pneumocystis jirovecii. The presentation of PCP in HIV-positive patients is well-known and consists of a triad of dyspnea, fever, and cough, whereas the presentation of PCP in HIV-negative patients is atypical and consists of a sudden outbreak, O-2 desaturation, and a rapid lethal outcome without therapy. Despite the availability of direct and indirect identification methods, the diagnosis of PCP remains difficult. The cycle threshold (C-T) values obtained by quantitative PCR (qPCR) allow estimation of the fungal burden. The more elevated that the fungal burden is, the higher the probability that the diagnosis is pneumonia. The purposes of the present study were to evaluate the C-T values to differentiate colonization and pneumonia in a population of immunocompromised patients overall and patients stratified on the basis of their HIV infection status. Testing of bronchoalveolar lavage (BAL) fluid samples from the whole population of qPCR-positive patients showed a mean C-T value for patients with PCP of 28 (95% confidence interval [CI], 26 to 30) and a mean C-T value for colonized patients of 35 (95% CI, 34 to 36) (P < 10(-3)). For the subgroup of HIV-positive patients, we demonstrated that a C-T value below 27 excluded colonization and a C-T value above 30 excluded PCP with a specificity of 100% and a sensitivity of 80%, respectively. In the subgroup of HIV-negative patients, we demonstrated that a C-T value below 31 excluded colonization and a C-T value above 35 excluded PCP with a specificity of 80% and a sensitivity of 80%, respectively. Thus, qPCR of BAL fluid samples is an important tool for the differentiation of colonization and pneumonia in P. jirovecii-infected immunocompromised patients and patients stratified on the basis of HIV infection status with different C-T values.