Mouse hepatoma cells were established in vitro as a permanently growing line designated Hepa. The mass population and a subclone were characterized for their karyotype and their retention of liver-specific properties. An examination of 17 hepatic traits revealed that the cell lines secreted several serum proteins. The activities of a number of liver-specific enzymes, however, appeared to be absent in these cells. The identification of differentiated properties of cultured hepatoma cells permits the use of these lines in a variety of studies such as cell hybridization, biochemical analysis of tissue-specific gene products, and the modulation of expression of genes governing differentiated phenotypes. This report presents the analysis of a broad spectrum of characteristics and thereby describes one of the most fully defined hepatoma cell lines of murine origin in the literature.