Nucleotide sequence analysis of a cDNA clone corresponding to the XBL-I open reading-frame of bovine leukemia virus (BLV) revealed that the AUG initiation codon was located 44 bases downstream from that of the env gene and was part of the p34x mRNA splice donor. . .ATGG/GTAA at the end of the pol gene sequence. RNA from this clone was synthesized in vitro by the SP6 RNA polymerase and translated into a 34,000 mol wt protein in rabbit reticulocyte lysates. The protein (p34x) is recognized in Western blots by most sera of BLV-infected sheep and tumor-bearing cattle, by an anti-synthetic peptide rabbit serum, and by the serum of a rabbit immunized by XBL-I RNA programmed reticulocyte lysates. Both sera react with a 34,000 mol wt protein present in nuclei of BLV-infected cells.