Increasing evidence suggests involvement of free-radical species in the development of oxidative DNA damage, the consequences of which have been implicated in a number of degenerative disorders associated with the aging process. Here we report the application of a single-cell gel electrophoresis (comet) assay for assessing levels of DNA damage in canine and feline leukocytes. Leukocytes were collected from 24 healthy adult cats and dogs and subjected to DNA damage ex vivo by exposure to a range of hydrogen peroxide (H(2)O(2)) concentrations (0-250 micromol/L). The optimal concentration of H(2)O(2) to induce a significant increase in DNA damage was 100 micromol/L for both canine and feline leukocyte samples. Levels of DNA damage were assessed and quantified by visual and computer image analysis. The results obtained showed high correlations between visual scoring and computer image analysis for feline samples (percentage DNA in tail, R(2) > 0.99; tail moment, R(2) > 0.95; tail length, R(2) > 0.90) and canine samples (percentage DNA in tail, R(2) > 0.97; tail moment, R(2) > 0.95; tail length, R(2) > 0.91). In conclusion, this method provides a way of assessing levels of DNA damage utilizing visual and/or computer image analysis in the feline and canine systems. With the capacity of the comet assay to be able to measure end products of free-radical reactions, it is a useful tool for determining the optimal effects of dietary antioxidants on a reliable biomarker of oxidative stress such as cellular DNA status in cats and dogs.