A double polymerase chain reaction (PCR) assay has been devised for the direct detection of bovine leukemia virus (BLV). The assay was directly performed on blood leukocytes, avoiding the DNA-purification procedures. The PCR products were identified by gel-electrophoresis and the specificity of the test was confirmed by hybridization with a biotinylated oligonucleotide probe. When testing the sensitivity of PCR, less than eight genome copies of the provirus were detected in the background of two million negative lymphocytes. In a BLV infected herd 22 animals of various age groups were examined by the indirect (serological) diagnostic tests of agar-gel immunodiffusion and indirect ELISA as well as by the direct detection method of PCR. The tests were repeated at monthly intervals on five occasions. When examining the specimens from cows and heifers, a close agreement was found between the results of the various methods. The newborn calves, which were the offspring of BLV infected mothers, were consequently negative in PCR throughout the experimental period. However, in the indirect tests the calves were positive during the first samplings and became negative only around four months of age. Since the indirect tests can not discriminate infection from colostral immunity, PCR proved to be a useful complementary assay for the safe diagnosis of BLV infection in young calves.