A newly developed experimental model system was used to determine in vitro transformation-specific parameters which correlate with tumorigenicity. The data suggested that clonal herpes simplex virus type 2-transformed syngeneic rat embryo cells with intermediate, transformed rat embryo fibroblasts (t-REF-G-1) or high, rat fibrosarcoma tumorigenic potential in syngeneic rats could be differentiated from clonal transformed nontumorigenic (t-REF-G-2) and nontransformed rat embryo fibroblast cells by their growth to increased saturation density and cloning efficiency in soft-agar medium. All clonal herpes simplex virus type 2-transformed cells, regardless of tumorigenic potential, possessed an increased rate of hexose transport and plasminogen activator activity and were less fibroblastoid in morphology compared to nontransformed rat embryo fibroblast cells. There were no significant difference in cell doubling time or total phospholipid composition between clonal transformed tumorigenic, nontumorigenic, and nontransformed cells.