Three double negative (BoCD4-, BoCD8-) bovine T cell lines, BTL-PC3, BLT2, and Pr2181, which have been established from bovine lymphosarcomas, were examined for expression and molecular function of bovine c-myb genes. BTL-PC3 expressed 4.0- and 3.6-kilobase c-myb transcripts, and BLT2 and Pr2181 expressed a 3.8-kilobase c-myb message. The c-Myb protein (75 kDa) was detected in Pr2181 but not as a clear band in BLT2, while BTL-PC3 exhibited a 65-kDa c-Myb band in immunoprecipitation tests with anti-Myb antiserum. Nucleotide sequences for c-myb cDNA clones from BTL-PC3 and BLT2 indicated that the predicted bovine wild-type c-Myb from BLT2 consists of 640 amino acids whereas that from BTL-PC3 consists of 555 amino acids lacking 85 internal amino acids. This deleted DNA region (255 base pairs consisting of 85 amino acids) corresponds to the human genomic exon 9 encoding a negative regulatory domain in the c-myb gene. Upon cotransfections with reporter plasmids containing myb binding sites, the internally deleted c-Myb exhibited a 3-fold higher transcriptional activity than the wild-type c-Myb in chloramphenicol acetyltransferase assays. These results indicate that internal DNA deletion in the c-myb gene is directly involved in the enhancement of transcriptional activation in bovine T lymphoma cells.