The hormone regulation of viruses has been of great interest since the discovery of glucocorticoid stimulation of mouse mammary tumor virus via a hormone response element in the viral long terminal repeat (LTR) promoter region. This report describes the investigation of the hormone responsiveness of bovine leukemia virus (BLV), an oncogenic retrovirus that infects dairy and beef cattle worldwide. It is a member of the human T cell leukemia (HTLV)/BLV group of retroviruses, which encode a protein, Tax, that is essential for regulating transcription of their own proviruses and for transforming host cells. We investigated the responsiveness of BLV to the hormones 17 beta-estradiol, progesterone, prolactin, insulin, and dexamethasone, a potent glucocorticoid. Only dexamethasone, in combination with insulin or insulin/prolactin, consistently stimulated BLV expression, as measured by reverse transcriptase activity, RNA blot hybridization (Northern blots), and CAT (chloramphenicol acetyltransferase) reporter assays of cell lines transiently or stably transfected with the BLV LTR. This effect required the presence of glucocorticoid receptors and Tax. This is the first report of hormone responsiveness in a virus of the HTLV/BLV group.