A sensitive method was developed for quantitation of N-nitrosomorpholine (NMOR) formation in vivo in rats. A major metabolite of NMOR, N-nitroso(2-hydroxyethyl)glycine, was detected by gas chromatography-thermal energy analysis in the urine of rats treated with trace levels of NMOR or treated sequentially with morpholine and sodium nitrite. Since significant levels of 2-hydroxyethylglycine do not seem to occur in rat urine, even after treatment with morpholine, artefact formation was not a problem. Formation of 1 micrograms NMOR in vivo was readily detected. The formation of NMOR from morpholine and sodium nitrite in vivo in rats appeared to depend on the doses of morpholine and [sodium nitrite]2 and occurred in higher yield than reported for formation of N-nitrosoproline, as expected. The formation of NMOR from morpholine and sodium nitrite under the conditions of the carcinogenicity study of Shank and Newberne was quantified. The daily levels of NMOR formation were highly variable, but the mean level was consistent with the tumorigenicity data in that dose-response study.