Mycoplasma bovis DNA was detected in lung tissue of experimentally infected calves by in situ hybridization (ISH) with a nonradioactive, digoxigenin-labeled DNA probe. The 171-base pair DNA probe targeting part of the gene of the major immunodominant variable surface protein A, which is conserved among all vsp genes, was generated by polymerase chain reaction. Four calves between 57 and 63 days old were inoculated intratracheally with 30 ml of a suspension of M. bovis strain 1067 containing 7 x 10(4) colony forming units per milliliter. Two calves inoculated with 30 ml of sterile medium served as control animals. The calves were euthanized and then examined 21 days after inoculation. The ISH method developed in the current study was suitable for the detection of M. bovis DNA in formalin-fixed, paraffin-embedded lung tissue and may be a valuable tool for diagnostic purposes and for further investigating the pathogenesis of M. bovis infection.