The capabilities of atmospheric pressure chemical ionization liquid chromatography/mass spectrometry (APCI-LC/MS) were investigated for the analysis of urinary 2,5-hexanedione (2,5-HD) and for the identification and characterization of other n-hexane Phase I metabolites in hydrolized urine samples. Chromatography was performed under reversed phase conditions at 0.75 mL min-1 flow rate. The ionization of 2,5-HD and other n-hexane metabolites was obtained in positive ion mode. After optimization of several interface parameters, the linearity, sensitivity and precision of the method were determined operating in the selected ion monitoring mode. Detection limits were 0.02 and 0.05 mg L-1 in water and urine respectively, with linear calibration curves in the 0.05-10 L-1 concentration range. Repeatability and both intra-day and inter-day precision were determined at two concentration levels (0.5 and 5.0 mg L-1), and relative standard deviations were in the 1.3%-5.3% range. The method was applied to the quantitative analysis of 2,5-HD in urine samples from an external Quality Assurance Programme for Organic Solvent Metabolites. Moreover, the metabolites 5-hydroxy-2-hexanone, 2,5-hexanediol and 4,5-dihydroxy-2-hexanone were identified and confirmed in hydrolyzed urine of rats exposed to n-hexane.