Enzootic bovine leukosis is caused by the bovine leukemia virus (BLV) and has a world wide distribution in cattle. Due to the program for eradication of BLV-infections in Germany the BLV incidence in cattle declined and only few new cases seem to occur per year. On the other hand, BLV-infected cattle with low, transient or without BLV-antibody titers are difficult to identify as BLV-infected. These animals may be sources for new infections. It was the aim of this study to compare the suitability of agargel-immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) for diagnosis of BLV-infected cattle. We investigated a herd with 10 cows, where after a long period when the herd was negative suddenly a positive serological reaction appeared. In addition 64 animals from 6 federal states of different herds with doubtful serological reactions found in previous tests were included. In the herd with 10 cows we were able to detect BLV-infection in one animal 8 weeks earlier with PCR than with ELISA. Investigation of 56 adult cattle and 3 calves from different herds with both PCR and ELISA showed that 51 animals were positive in ELISA and 55 in PCR. Seven animal were positive in PCR and negative in ELISA. Three calves yielded negative results in PCR and positive results in ELISA. One cow which was positive in previous serological tests was negative in ELISA, AGID and PCR. Restriction fragment length polymorphism analysis demonstrated that the majority of the cattle was infected with the same BLV provirus variant. The four PCR variants used in this study yielded a similar sensitivity for BLV provirus detection. In conclusion, compared to the serological tests, PCR detects BLV-infection earlier in naturally infected cattle. The method is also a useful tool to exclude or confirm BLV-infection in cattle with doubtful serological results. PCR may be used to complement the serological tests in the diagnosis of BLV-infection.