To be informative for chemoprevention, animal models must both closely emulate human disease and possess surrogate endpoint biomarkers that facilitate rapid drug screening. This study elucidated site-specific histopathological and biochemical surrogate endpoint biomarkers of spontaneous epidermal carcinogenesis in K14-HPV16 transgenic mice and demonstrated that the incidence and severity of these markers were decreased by the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO). The cumulative incidence of visible epidermal cancers in 127 untreated transgenic mice was 42% by 52 weeks of age, most frequently affecting the chest as flat lesions in association with chronic ulcers, or in the ear as protuberant masses. Microscopic malignancies were detected in 39% of 32-week-old transgenic mice and were found to emerge from precursor lesions that were of two distinct types: dysplastic sessile ear papillomas and hyperproliferative follicular/interfollicular chest dysplasias. ODC activity and tissue polyamine contents were differentially elevated in ear and chest skin during carcinogenesis, such that there was a marked elevation of both parameters of polyamine metabolism as early as 4 weeks of age in the ear, whereas in the chest, polyamine metabolism was increased significantly only in the late stages of neoplastic progression and in epidermal cancers. Administration of 1.0% DFMO in the drinking water from 4 to 32 weeks of age prevented both visible and microscopic malignancies and significantly decreased the incidence of chest and ear precursor lesions. ODC activity and tissue putrescine content were markedly diminished by DFMO chemoprevention in ear skin, whereas there was a more modest decline of these parameters in chest skin. DFMO treatment of transgenic mice from 28 to 32 weeks of age was associated with an absence of ear cancer and a marked regression of dysplastic papillomas. In contrast, the results in chest skin were complex in that the severity of chest precursors diminished, but their incidence was unchanged, and microscopic cancers were still detectable within these lesions. Collectively, this study highlights the utility of multistage epidermal carcinogenesis in K14-HPV16 transgenic mice both for the study of the biology of, and as a screening tool for, novel drugs and chemopreventive regimens.