We describe the detection and characterization of an activated c-N-ras allele from a gamma-radiation-induced canine acute nonlymphocytic leukemia (ANLL). The activated allele was detected by use of the NIH3T3 transfection/transformation assay. The leukemia DNA had a transforming activity of 0.0125 foci/microgram. By the use of a double anti-ras antibody enzyme-linked immunoblot assay, we have dissected the lesion within the activated c-N-ras allele. Aspartic acid has been substituted for the normal glycine at position 12 in the activated p21c-N-ras. The expression of the mutant p21ras has also been detected in an in vivo passage of the radiation-induced canine ANLL from which the activated c-N-ras allele was isolated. We have demonstrated sufficient homology between canine c-N-ras genes and the human cDNA c-N-ras clone, p6a1, that allows this probe to be used in Southern blotting of canine tissues. In addition, anti-ras antibodies generated against both murine and human ras antigens are capable of detecting canine p21ras species.