Up to now, the carcinogenicity of a substance, i.e., its cancer-causing effect, has been usually determined with the help of extensive animal testing. The in ovo carcinogenicity assay (IOCA) has been suggested as a rapid and inexpensive, non-animal, method for carcinogenicity testing and for experimental studies on mechanisms of carcinogenesis. The substance to be tested is injected into a fertilized ovum. No later than four days before the hatching date, the embryos are released, and the liver is removed for identifying the effect of hepatocarcinogens. Histological, cytological and molecular biological alterations of the embryonic liver have been induced with a variety of chemical carcinogens. After sufficiently high doses of hepatocarcinogens, tubular structures predominate in the liver and replace the normal trabecular pattern. The cell and nuclear size of the hepatocytes in embryonic liver is severely increased after exposure to chemical carcinogens over a wide dose range including doses that fail to elicit cytotoxicity in the embryonic liver. The in ovo exposure of avian embryos to hepatocarcinogens has also been shown to induced focal lesions in the embryonic livers. In embryonic turkey liver, in ovo exposure to carcinogens mostly induced basophilic hepatocellular altered foci (foci of altered hepatocytes) that were similar to carcinogen-induced lesions in rodent liver. In embryonic quail livers, most of the focal lesions were composed of cells that morphologically appeared more similar to cholangiocytes than to hepatocytes. The in ovo exposure of embryonic liver to hepatocarcinogens resulted in damage to the mitochondrial DNA (mtDNA). Electrophoresis on agarose gels revealed a carcinogen-induced effect on the molecular size of the mtDNA. The content of mtDNA of the regular size of 16 kilobases (kb) dose-dependently decreased. At the same time, the amount of fragmented mtDNA of various sizes increased. The advantages of bird embryos for carcinogenicity testing are considerable: The IOCA is rapid, since the in ovo phase does not exceed 24 days. Since no facilities for animal housing are required, it is less expensive than whole animal experiments. There is less potential of human exposure during the experiment than in animal experiments and studies in a non-rodent system may help to discriminate between chemicals that are rodent specific carcinogens and chemicals that may constitute a potential cancer hazard to man.