Expression of bovine leukemia virus (BLV) has been considered to be blocked at the transcriptional level in vivo, since viral RNA species are not readily detected in freshly isolated leukocytes from BLV-infected animals. However, the presence of a persistent antiviral antibody response in infected animals suggests that some degree of virus expression must occur in vivo. The purpose of this study was to determine whether BLV RNA species could be detected by using the polymerase chain reaction in normal or neoplastic lymphoid cells freshly isolated from naturally or experimentally BLV-infected cattle and sheep, respectively. Primers designed to detect a 2.1-kb doubly spliced BLV tax/rex-specific mRNA were used to amplify cDNA copies of RNA derived from infected animals. The amplified viral product was then detected with a radiolabeled BLV tax/rex-specific probe. BLV-specific RNA was detected readily in freshly isolated peripheral blood leukocytes derived from BLV-seropositive cattle or sheep with persistent lymphocytosis and less readily in peripheral blood leukocytes from BLV-seropositive but hematologically normal animals. BLV-specific RNA was also detected in fresh samples of BLV-induced lymphosarcomas. Normal and neoplastic lymphoid cells from BLV-seronegative animals were uniformly negative under similar conditions. These primers also amplified the same viral product from genomic DNA derived from BLV-seropositive animals, providing further evidence for in vivo transcription and suggesting that BLV RNA-dependent DNA polymerase is capable of reverse transcribing the 2.1-kb mRNA in vivo. The demonstration of transcriptional products of BLV in vivo proves that viral latency in BLV infection is incomplete.