Intravenous vaccination of rats with either viable or Formalin-inactivated Mycoplasma pulmonis reduced the incidence and severity of lower respiratory tract lesions after intranasal challenge with viable organisms. Intranasal vaccination with killed organisms reduced the severity of rhinitis, but did not affect lesions in any other region of the respiratory tract. The maximum protection against upper tract lesions (rhinitis, otitis, and laryngotracheitis) was provided by intravenous immunization with viable organisms. Dual vaccination (intraperitoneal plus intranasal) with killed organisms provided no significant protection in any segment of the tract. However, these ineffective vaccine regimens did not potentiate the lesions. These results conclusively demonstrate that vaccination of rats against mycoplasma respiratory disease is feasible and also suggest that systemic vaccination may provide greater protection for the lungs than intranasal vaccination, at least when equivalent antigen doses are used.