This study was planned to evaluate the possible application of PCR as a rapid method for diagnosis of PCP in children with haematological malignancies under immunosuppressive therapy by comparing PCR- based assay with the conventionally- used methods for diagnosing PCP which include direct staining of clinical samples or immunofluorescence (IF) assay. This study was conducted on 50 patients with haematological malignancies. They were chosen among the in-patients children of the Pediatric Oncology Unit of Kasr El-Aini Centre for Oncology, Faculty of Medicine, Cairo University. These children were on chemotherapy for pediatric haematological malignancies. The children were also suffering of neutropenic fever and associated respiratory tract manifestations which did not respond to first and second lineantibiotic therapy. Clinical samples (bronchoalveolar lavage, sputum or nasopharyngeal aspirate) were collected from patients for detection of P. carinii. The samples were examined by direct film (stained by Giemsa staining), immunofluorescence, single and nested PCR. Number of positive cases to P. carinii detected by all methods was 36 out of 50 patients (72%), while those detected by IF only were 4 out of 30 patients (13%). The number and percentage of cases tested positive to P.carinii detected by all methods showed slight difference among differentsampling methods (71%, 75% and 67% tested positive from NPA, sputum and BAL samples, respectively). IF was chosen as gold standard to estimate diagnostic values of the other methods used to confirm PCP. Very high sensitivity and NPV were recorded for nested PCR (100%). However, nested PCR showed the lowest specificity and PPV. Single PCR and Giemsa stain showed 100% specificity and PPV (100%).Sensitivity of Giemsa stain was, however, extremely low (25%). In conclusion, IF stain, is more sensitive than Giemsa stain and almost as sensitive as single PCR.Single PCR can be applied for the diagnosis of pneumocystis infection in immunosuppressed patients. Nested PCR may be of value for asymptomatic immunosuppressed patients identifying those at high risk of developing PCP in the future.