More than 10 kinds of heterocyclic amines (HCAs), showing mutagenic and carcinogenic potency, have been isolated from cooked fish and meat. But many researchers say that the contribution ratio of HCAs to human cancer is very low. Our purpose in this experiment was to investigate the possibility of the formation of HCAs under moderate conditions, including in vivo. A mixture of d-glucose, creatinine, and amino acid such as glycine, methionine, threonine, and proline was dissolved in phosphate-buffered solution (pH7.4) and incubated at 37 degrees C, 50 degrees C, 128 degrees C. At an appropriate time, an aliquot of the reaction solution was treated with blue cotton. HCAs were separated from the blue cotton by elution with 2% ammoniacal methanol. The eluates were submitted to the Ames test, the micronucleus test for determination of mutagenicity, and also LC-MS analysis for the detection of HCAs. Nonadsorbates to blue cotton were treated with dichloromethane and then subjected to the mutagenicity test. In the Ames test, the mutagenic activity of the reaction mixture increased with an increase of the reaction temperature. The HCA fraction from 50 degrees C incubated solution showed high frequency in the micronucleus test using HepG2 cells. The dichloromethane fractions contained other type of mutagens different from HCAs. In HCA fractions, IQ, MeIQx, 4,8-DiMeIQx, and 7,8-DiMeIQ were identified. It is said that the heating process is an essential factor in the formation of HCAs. But our experiment shows that HCAs are produced not only in the cooking process, but also in moderate conditions such as 37 degrees C and 50 degrees C.