The first temperature-sensitive mutants of epithelial cells transformed with chemical carcinogens have been isolated. Like the wild-type transformed parental cells, the mutants readily grow in agar suspension at 36 degrees, but in contrast to the wild type, they do not do so at 40 degrees. Detailed studies of one of these mutants, TS-223, indicate that at high temperature it also has reduced cloning efficiency in monolayer culture and a lower saturation density. Scanning electron microscopy revealed that at 40 degrees confluent cultures of TS-223 consist of a monolayer of generally flat polygonal cells, whereas 36 degrees cultures contain many patches of piled-up cells that are spherical and have rougher surface membranes. All of these cellular changes are reversible with upward or downward temperature shifts. The temperature-sensitive lesion appears to reside in a host cell gene which modulates expression of the transformed cell phenotype. These mutants may provide a useful system for elucidating the minimal biochemical changes required for expression of the transformed phenotype in epithelial cells.