The reaction of 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) with non-radical biological components produced spin adducts with ESR signals. The reactions of DBNBS with Trp, Gly-Trp, Trp-Gly, Pro, Cys and glutathione at pH 7.5 and room temperature for more than 1 hour gave the nitroxyl free radicals with ESR signals, whereas the reactions with other amino acids and bovine serum albumin did not. Among the amino acids and the peptides, Trp and Trp-containing peptides gave the most intense signals. The reactions of DBNBS with unsaturated fatty acids,i.e., linoleic acid and oleic acid, gave weak ESR signals, whereas the reaction with stearic acid did not. While DBNBS gave no ESR signals by the reactions with DNA, nucleosides and nucleobases, it caused strand breaking in supercoiled DNA. DBNBS also gave ESR signals by the reaction with human plasma similar to those from the reaction with Trp. It was suggested that the nitroxyl free radicals were produced by the addition of DBNBS to the amino acids and unsaturated fatty acids followed by oxidation in the presence of DBNBS. Hence, the use of DBNBS spin trap to detect free radicals in systems containing these biological components after long incubation may give misleading results.