The detection of BK polyomavirus (BK virus, BKV) in kidney tissue is hampered by nonspecificity of antibodies suited to immunohistochemistry, and nonspecific background with in situ hybridization. The biotin-labeled DNA probe that is commercially available (Enzo Life Sciences, Inc.) shows good signal, but the intrinsic background in kidney tissue is high. We determined that the intrinsic background is due to endogenous biotin or biotin-binding activity in the renal tubular epithelium. Neither antibody blocking procedures nor an avidin/biotin block were entirely satisfactory for eliminating this background staining. We developed a digoxigenin-labeled DNA probe, and protocol, for detecting BK virus in formalin-fixed, paraffin embedded, kidney tissue obtained at autopsy. The hybridization signal is strong and there is no perceptible background staining. Eleven negative control kidneys all failed to hybridize. Conditions for low stringency hybridization may be employed, detecting both the related JC polyomavirus and BKV. Alternatively, high stringency hybridization conditions may be utilized, detecting BKV only. BK associated tubular necrosis is clearly demonstrated in two cases of BK nephritis.