Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the lack of an infectious molecular clone and the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in North America, and only one full-length sequence is available for each of ENTV-1 and ENTV-2. In order to understand the molecular evolution of ENTV-1, the full-length genome sequence of ten ENTV-1 proviruses derived from clinical samples of ENA isolated from conventionally reared sheep in Canada and the United States was determined. The North American ENTV-1 (ENTV-1(NA)) genomes shared greater than 96% sequence identity with the European ENTV-1 sequence (ENTV-1(EU)). Most of the amino acid differences were found in Orf-x, which in the corresponding ENTV-1(EU) genome is truncated by 44 amino acids. Apart from Orf-x, the long terminal repeat (LTR) is where the majority of differences between ENTV-1(NA) and ENTV-1(EU) reside. Overall, there was an unusually high degree of amino acid conservation among the isolates suggesting that ENTV-1 is under stabilizing selection and K(a)/K(s) ratios calculated for each of the viral genes support this hypothesis. The unusually high degree of genetic stability of the ENTV-1 genome enabled us to develop a hemi-nested PCR assay for detection of ENTV-1 in clinical samples. Additionally, multiple nasal tumor cell clones were established and while most had lost the provirus by passage 5; one polyclonal line retained the provirus and attempts are being made to culture these cells. These tumor cells, the first of their kind, may provide a system for studying ENTV-1 in vitro. This work represents an important step in the study of ENTV and sets the foundation for the construction of an infectious molecular clone of ENTV-1.