Using single primer pairs Y3 and Y4 in situ polymerase chain reaction (in situ PCR) was successfully performed on the specimen slides of peripheral leukocytes. By both of the direct digoxiginin-11-dUTP incorporation into PCR products with in situ PCR (direct in situ PCR) and in situ PCR followed by detection of in situ hybridization (indirect in situ PCR), DNA fragments specific for human Y chromosome were obviously amplified in cellular nuclei of specimens on the slides. The results were verified by Southern analysis. The methodology of in situ PCR and its application were discussed.