Seventy anti-rabies virus monoclonal antibodies (Mabs) were tested for reactivity with rabies and rabies-related viruses in formalin-fixed (FF) tissues. Forty-three of the Mabs were directed against the glycoprotein and 27 were directed against the nucleocapsid as determined by enzyme immunoassays and neutralization tests. Twenty of the anti-glycoprotein Mabs and one of the anti-nucleocapsid Mabs reacted with the rabies challenge virus strain (CVS) in FF tissue. These 21 Mabs were screened against other lyssaviruses in FF tissues: five rabies virus strains (coyote, skunk, raccoon, red bat, and silver-haired bat), and four rabies-related viruses (Australian bat lyssavirus, Duvenhage virus, Lagos bat virus, and Mokola virus). One of the anti-glycoprotein Mabs was reactive with all the virus strains screened. Another of the anti-glycoprotein Mabs reacted with all of the rabies virus strains tested, but not with any of the rabies-related virus strains tested. The remaining Mabs had reactivity patterns that could be useful for characterizing lyssaviruses in FF tissues.