The 60-70S RNA of Mason-Pfizer virus (MPV) was iodinated in vitro and used in both direct and competitive molecular hybridization studies. MPV proviral sequences are present at a frequency of approximately one to two copies per haploid genome in the DNA of experimentally infected human cells. By nucleic acid competition hybridization, MPV RNA was found to be indistinguishable from the RNA of a virus (X381) isolated from a rhesus mammary gland and from RNA isolated from the cytoplasm of AO cells (Parks et al., 1973) and HeLa cells (Gelderblom et al., 1974), both previously reported to produce MPV-related particles. No homology was observed, however, between MPV RNA and the RNA, or the DNA, from two clones of HeLa cells obtained from the American Type Culture Collection. Hybridization of MPV 60-70S RNA to the DNA of normal tissues of humans and to the DNA of 11 other species revealed that MPV is not an endogenous virus of any of these species. Competition hybridization revealed no detectable sequence homology between the RNA of MPV and the RNAs of simian sarcoma virus, murine mammary tumor virus, murine leukemia virus, BUdR-induced guinea pig virus, or avian myeloblastosis virus. These nucleic acid studies substantiate previous ultrastructural and immunological findings that MPV and morphologically similar isolates constitute a distinct group of oncornavirus.