N,N-dimethylformamide (DMF) is a solvent widely used to prepare synthetic fibers. Biomonitoring of DMF is usually performed by measuring urinary N-methylformamide, which allows us to estimate exposure during the working day. An alternative biomarker is the mercapturic acid N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) whose excretion accounts for about 13% of the absorbed DMF dose. Owing to its slow excretion (mean half-life = 23 hours) the urinary levels of AMCC at the end of a workweek reflect the cumulative dose of DMF during the whole week. Methods given in literature for measuring AMCC need the derivatization of the molecule before analysis. The paper describes a method for the determination of urinary AMCC by high-performance liquid chromatography (HPLC) with direct UV detection. Samples were purified by solid phase extraction with C18 and ENV+ cartridges, then 10 microliters were directly injected onto an Aminex HPX-87H Ion Exclusion column maintained at a temperature of 37 degrees C. Analyses were performed by isocratic run with 1 mM sulphuric acid delivered at 0.85 mL/min. The detector was set at 196 nm. Under these conditions, AMCC eluted at 11.1 min., and the detection and quantification limits were 1.32 mg/L and 3.96 mg/L, respectively. The performance of the method was evaluated on samples containing 25 mg/L and 400 mg/L of AMCC: each sample was analysed three times. The mean recovery of the extraction procedure was 88.3%. The precision (CV%) and the accuracy (Error%) ranged from 0.8% to 2.9%, and from -1.2% to +3.2%. The calibration curve was linear up to a concentration of 1000 mg/L, the coefficient of correlation was r = 0.9997. AMCC was measured in urine samples from 30 exposed and 20 unexposed (smokers and nonsmokers) subjects. Measurable amounts of AMCC were found in all of the samples from workers exposed to DMF; on the contrary, none of the samples from unexposed subjects contained this metabolite. The proposed method is sufficiently sensitive and specific for the evaluation of occupational exposure to DMF, thus it could be useful for the biological monitoring of workers exposed to this solvent.