The disposition and the metabolism of 2-chloroethyl nitrosocarbamoylcystamine (CNCC), a new antitumor agent, has been studied in rats. For this purpose, three separate labeled species of CNCC have been used. The tissue distribution and the elimination of the radioactivity were determined in animals after gavage with a single dose of each labeled species of CNCC (35 mumol/kg). It was observed, after analysis of plasma taken at timed intervals after administration, that little radioactivity co-chromatographed with the parent compound. These data suggest that CNCC undergoes an important first-pass metabolism, but chromatographic analysis provided evidence for the formation of four main metabolites. These biotransformation products were isolated from pooled plasma extracts of rats treated with 200 mumol/kg of unlabeled CNCC. They were identified by the combined use of mass spectrometry and chromatographic properties. These metabolites are sulfinyl and sulfonyl derivatives arising from the bioreduction of the disulfur bridge of CNCC with subsequent methylation and oxidation. These compounds are potentially active cytostatic agents. The evaluation of their antitumor activity is currently under investigation.