Hormones play an important role in the bodies of mammals because they act on metabolism and reproduction, as well as coordinate and regulate activities of different parts of the body, organs and tissues. Steroid hormones possess the capacity to increase weight gain and to reduce the feed conversion ratio, therefore they can be used for anabolic purposes in animal fattening. Taking into account the toxicity of hormones, particularly their cancerogenic and mutagenic properties, the use of this group of compounds for animal fattening purposes has been prohibited in the European Union, but natural hormones are allowed in some countries, for example USA and Australia. However, steroid hormones are also natural constituents of muscle and fatty tissues as well as of livers and kidneys of nontreated animals. Their occurrence is not restricted to mammals. Steroid hormones have also been determined in fish and poultry; they have been found in other animal-derived food such as milk and eggs. The residues of anabolic hormones in animal tissue as a result of implantation or illegal use can be a hazard for humans. Therefore the monitoring of hormone residues in biological samples of animal origin is mandatory. Several cases exceeding the acceptable limits of 17β-estradiol and testosterone in bovine serum were noted in Poland in the last five years. Moreover, the presence of endogenous 19-nortestosterone in the urine of pigs was observed and no residues of synthetic hormones in tissues of slaughter animals were found.