The 1,3-diethyl-2-thiobarbituric acid (DETBA) assay for nicotine metabolites has been improved so that it can be used to determine the concentrations of nicotine and up to 12 metabolites in the urine of humans and laboratory animals, including phase 2 metabolites. The products of beta-glucuronidase cleavage found in human urine were mainly trans-3'-hydroxycotinine, cotinine, and a small amount of nicotine. Following isolation, spectroscopic analyses showed the structure of the nicotine DETBA derivative to be the one-to-one ring-opening product of DEBTA and the cyanopyridinium salt of nicotine.