Abstract A fluorigenic high performance liquid chromatographic (HPLC) assay for the determination of acetaldehyde in plasma and hemoglobin-associated acetaldehyde is described. The assay is based on the reaction of acetaldehyde with two molecules of 1,3-cyclohexanedione in the presence of ammonium ion to form a fluorescent species followed by reverse phase HPLC separation of specific aldehyde derived compounds. The assay is specific; and has sensitivity in the picomole range, with intraassay precision of less that 3.5% and interassay precision of less than 15%. The total run time is less than 6 minutes on HPLC. Hemoglobin-associated acetaldehyde levels were higher than the levels found in plasma. Endogenous levels of acetaldehyde in samples obtained from teetotalers were found to be 0.43±0.04 (S.D.) μM in plasma and 74.2±16.1 nmol/g protein as hemoglobin-associated acetaldehyde. The levels of both plasma acetaldehyde and hemoglobin-associated acetaldehyde were significantly higher in patients reporting to a center for alcohol treatment than the levels encountered in teetotalers.