An in situ hybridization (ISH) technique using a digoxigenin (DIG)-labeled cDNA probe detected avian reovirus (ARV) RNA in formalin-fixed, paraffin-embedded chicken tissues. Tissues were collected 3 and 10 days following inoculation with the R-2 or the S1133 strain of ARV. The cDNA clone HJp1, located on the S1 gene segment of the ARV S1133 strain, was used to prepare a nonradioactive probe. The ISH assay localized ARV RNA in infected tissues including heart, liver, intestine, pancreas, and tendon. No positive-stained cells occurred in sections from uninfected chickens.