Axonal damage has been highlighted recently as a cause of neurological disability in various demyelinating diseases, including multiple sclerosis, either as a primary pathological change or secondary due to myelin loss. To characterize and quantify axonal damage and loss in canine distemper demyelinating leukoencephalomyelitis (DL), formalin-fixed paraffin-embedded cerebella were investigated histochemically and immunohistochemically using the modified Bielschowsky's silver stain as well as antibodies against nonphosphorylated (n-NF), phosphorylated neurofilament (p-NF) and β-amyloid precursor protein (β-APP). Injured axons characterized by immunoreactivity against n-NF and β-APP were detected in early distemper lesions without demyelination. In subacute and chronic demyelinating lesions the number of injured axons increased. Moreover, a significant decrease in axonal density was observed within lesions and in the normal appearing white matter in DL as determined by morphometric analyses using Bielschowsky's silver stain and p-NF immunohistochemistry. Summarized, the observed findings indicate that axonal damage (i) occurs early in DL; (ii) can be detected before myelin loss; and (iii) represents a pivotal feature in advanced lesions. It must be postulated that axonal damage plays an important role in the initial phase as a primary event and during progression of nervous distemper as a result of demyelination.