Mycoplasma bovis is one of the most pathogenic of the bovine mycoplasmas. 7,8 It can cause mastitis, respiratory disease, arthritis, urogenital infections, and possibly keratoconjunctivitis and was first isolated from a mastitis case in 1961. Mycoplasmas require 1-2 weeks for culture and are easily overgrown by contaminants, even using appropriate media and incubation conditions. Serum antibody titers used to diagnose M. bovis infection can be misleading because the organism produces low serum antibody titers following respiratory infections. Because of lengthy culture and identification procedures, a rapid and easier method of detection and identification would be beneficial. DNA probes for many species of mycoplasmas have been developed. 5,8,12,15,16 Recently, the detection of M. bovis using an oligonucleotide probe was reported. 10 This article describes the development of a genomic DNA probe for detection of M. bovis. Mycoplasma bovis MBAT6 and M. bovirhinis 260-4; a Ureaplasma diversum, M. arginini, and Acholeplasma laidlawii; Escherichia coli, Klebsiella, Enterobacter, Staphylococcus aureus, and Streptococcus sp. (isolated from mastitis cases); Pasteurella multocida and P. haemolytica (respiratory tract isolates) and Proteus, Bacillus, and Pseudomonas (isolated from tissue samples at the Ohio Agricultural Research and Development Center [OARDC], Wooster, OH) were used in this study. Standard Frey medium and American Type Culture Collection (ATCC) medium #243 (700 ml 25 g/liter heart infusion broth, 200 ml 20% [w/v] inactivated horse serum, 100 ml 2.5% [w/v] yeast extract) were used to grow M. bovis, M. bovirhinis, M. arginini, and A. laidlawii. Ureaplasma diversum was grown in special medium containing 3.0 g mycoplasma broth base, 140 ml water, 0.8 ml 0.5% (w/v) phenol red, 40 ml horse serum, 20 ml 25% (w/v) yeast extract (pH 6.0), 4 ml 1.0% (w/v) urea, and 4 ml penicillin (100,000 units/ml). All mycoplasmas were grown at 37 C with increased CO, (7-10%) using CO, gas pack bags except U. diversum which required anaerobic conditions produced using an anaerobic gas pack. Bacteria were grown at 37 C on blood agar and brain-heart infusion broth with 10% horse serum added for Pasteurella multocida and P. haemolytica. Mycoplasma bovis broth cultures (200-500 ml) were inoculated and grown for 3 days and pelleted at 14,000 x g for 15 minutes at 4 C. The cell pellet was washed in TE buffer (10 mM Tris HC1 and 1 mM ethylenediaminetetraacetic acid; pH 7.5) and centrifuged at 14,000 x g for 15 minutes at 4 C. Pellets were resuspended in 5.0 ml TE buffer, 1.0 mg proteinase K was added, and the mixture was incubated for 1 hour at 37 C. Following incubation, 1.0 ml of lysis solution (5.0 g sodium dodecyl sulfate (SDS) in 50 ml water and 50 ml 100% EtOH) was added to the mixture and shaken vig-