AbstractThe three 2‐mercaptoethanol (ME) thioether conjugates of 2‐hydroxyacetanilide (2HAA) are detected and identified using liquid chromatography/electrochemistry (LC/EC). The conjugates are produced either by incubations of 2HAA with liver microsomes in the presence of NADPH and ME or by chemically oxidizing a solution of 2HAA and allowing it to react with ME. Identification of the three isomers is based on retention time and comparison of the electrochemical behavior of model compounds.Production of the three isomers by microsomal protein occurs only in the presence of NADPH and shows that 2HAA proceeds through a cytochrome P‐450 mediated pathway to a reactive intermediate similar to acetaminophen, possiblyN‐acetyl‐o‐quinoneimine.The relative percentages of each isomer produced were calculated as the percentage of the total peak areas of the isomers. Compound A (2HAA‐5‐ME) is produced in 53.7% and 24.6%, compound B (2HAA‐4‐ME) is produced in 16.2% and 70.3% and compound C (2HAA‐3‐ME) is produced in 30.1% and 5.1% by microsomal incubations and chemical