Abstract 2-Acetylaminofluorene, a well-documented carcinogen, is used extensively as a positive control in the bioassay of numerous suspect carcinogens. Consequently, there is a need for a rapid, sensitive analytical method for determining 2-acetylaminofluorene and its metabolites in urine. Such a method would be useful in monitoring workers for potential exposure as well as for determining the applicability of the test species by metabolism studies. A high pressure liquid chromatography method for the identification and quantitation of 2-acetylaminofluorene and its major metabolites in urine has been developed. The primary obstacle in developing the method was the presence of large amounts of endogenous urinary components in ether extracts of urine having chromatographic properties similar to 2-acetylaminofluorene and its metabolites. This problem was obviated by the use of a combination of Carbowax 400 and C 18/Corasil columns. N-Hydroxy-2-acetylaminofluorene was determined directly from the Carbowax 400 column. Fractions containing 2-acetylaminofluorene and its 1-, 3-, 5-, and 7-hydroxy metabolites were then separated from interfering materials and quantitated by fractionation on a C 18/Corasil column. The response of the system was linear down to 100 ng of N-hydroxy-2-acetylaminofluorene and to 10 ng of 2-acetylaminofluorene and the other metabolites.