Studies, including cytogenetic, immunofluorescence, infectivity, and tumorigenicity, were conducted to characterize 7 sublines of transformed rhesus foreskin cells chronically infected with Mason-Pfizer monkey virus (MPMV). These cell lines, referred to as MPfsl, released infectious MPMV and contained antigens common to MPMV-infected human lymphoblast (NC-37) and nontransformed MPMV-infected rhesus foreskin cells. Several of the MPfsl lines exhibited chromosomal changes typical of transformed cells and released interferon-like substances into the culture medium. MPfsl cells inoculated into newborn rhesus monkeys induced palpable nodules of donor cell origin at the sites of inoculation. Although these nodules were transient, explant cultures derived from the nodule biopsy specimens continued to release infectious virus; when subsequently reinoculated into other monkeys, these cultures induced nodules. These findings provide a convenient in vitro system for the study of MPMV and further define the identity of MPMV as an oncornavirus.