Equine papillomaviruses (EqPV) from naturally occurring cases of cutaneous papillomatosis in several ponies and one horse were isolated, cloned, and characterized. Group specific papillomavirus structural antigens were detected in sections of the papillomas by the peroxidase-antiperoxidase technique, and virions were observed in the in the nuclei of cells in the stratum granulosum and corneum. Negatively stained virions purified from papilloma homogenates by isopycnic CsCl centrifugation were 55 nm in diameter and had typical papillomavirus morphology. The entire viral genomes of two separate isolates were cloned at a single BamHI site into pBR322. A detailed restriction map of the viral genome is presented. Using nick-translated subgenomic fragments of BPV-1 as probes in Southern blot hybridizations, the organization of the EqPV genome was established. Southern blot analysis under various conditions of stringency revealed that EqPV shares relatively more homology with the late region of the BPV-1 genome and with the E2 region of the HPV-1 genome than with other parts of the same viral DNAs. Papillomavirus-specific sequences were found in papillomas from other anatomic sites using the EqPV DNA as a probe in Southern blot hybridizations. Genomes detected in DNA from penile papillomas had a different restriction pattern and hybridized to the EqPV probe only under nonstringent conditions.