Objective. To develop a possible alternative laboratory diagnostic method, an in situ polymerase chain reaction (ISPCR) for definitive diagnosis of human cytomegalovirus (HCMV) pneumonia in infants. Methods. The material involved in this study was formalin-fixed paraffin-embedded lung tissue specimens from 6 infants died of histopathologically diagnosed cytomegalic inclusion body pneumonia (CMIBP) and from 4 infants died of other diseases. When DNA extracts from the specimens of these 10 infants were tested by using a previously applied conventional PCR, the specimens from the 6 infants with CMIBP were positive for HCMV DNA, while those from the other 4 infants were negative. In the ISPCR, biotin-11-dUTP was used for labeling the amplified products and streptavidin, biotin-alkaline phosphatase for in situ color development. Results. The results of the study showed specific staining for HCMV DNA in the lung tissue slices from the 6 cases with CMIBP, while no staining was found in the specimens from the 4 infants who died of other diseases. The control ISPCR tests on the HCMV DNA positive specimens without adding either biotin-11-dUTP or Tag DNA polymerase or the primers for HCMV DNA showed negative results. The specific staining for HCMV DNA was seen in bronchial and alveolar epithelial cells, small vascular endothelial cells and leukocytes in the lumens of small blood vessels. Conclusions. The results suggested that the ISPCR was able to demonstrate both presence of HCMV DNA in the lung tissues and active HCMV infection; therefore the ISPCR may be applicable in definitive diagnosis of HCMV pneumonia when proper specimens such as lung biopsy material or bronchoalveolar lavage is available for the test.