In vitro assay for carcinogenesis, using mammalian cells, provide opportunity for rapid and inexpensive means, compared to in vivo assays, for studying carcinogenesis and for identifying potential carcinogens. These assays must, however, be shown to be reproducible, reliable and able to detect a variety of known carcinogens before they can be recommended for general use. We have, independently, reproduced a transformation assay which utilizes murine leukemia virus-infected rat embryo cells as targets. In the process a new culture, designated 2FR4(50), was generated to replace the F1706 line, of Freeman, which is no longer available. Through careful control of the assay parameters a readily reproducible test has been developed. In 2-4 culture passages after chemical treatment, morphologically transformed foci of cells are observed while no such foci are found in noncarcinogen treated or control cultures. Over 75 compounds have been tested in this assay; 20 of these are detailed here as representative of the chemically diverse types of carcinogens detected.