Frying of ground beef at 250 degrees C results in the formation of a series of mutagenic heterocyclic amines, possibly as many as 10 distinct compounds. In this study, the mutagens are separated by aqueous/acid extraction from the beef, XAD adsorption, acid/neutral/base-liquid/liquid extraction, preparative reverse phase h.p.l.c., normal phase h.p.l.c., and analytical reverse phase h.p.l.c. Identification is by low and high resolution mass spectrometry, u.v. absorption spectroscopy and nitrite sensitivity assays. More than 30% of the mutagenic material is identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). The previously described beef mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) contributes less than 12% of the total mutagenicity at high temperatures (300 degrees C) and less than 4% at lower temperatures. In addition to MeIQx major mutagens are seen with molecular ions at m/z 227 (C12H13N5), 209 (C13H11N3), and 176 (C9H12N4). Two very polar and two non-polar peaks making a relatively minor contribution to the total mutagenicity (less than 16% total) have not yet been identified. Estimates of the mass of these mutagens in the original cooked beef were made from the mass spectral analysis and the mutagenic recoveries. From one kilogram of cooked ground beef, 1.0 micrograms MeIQx, 0.02 micrograms of IQ, and up to 1.5 micrograms of additional mutagens are formed.