Molecular mechanisms of ethanol interaction with proteins are not well-understood. In the present study, direct interaction of ethanol with hydrophobic binding sites on fatty acid free bovine serum albumin (BSA) was determined using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), cis-parinaric acid, and 13C NMR. The affinity of ethanol for BSA (Kd) was (5.21 ± 0.31) × 10-2 mol. Ethanol (25-200 mmol) competitively inhibited 1,8-ANS binding to BSA in a concentration-dependent manner with a Ki (concentration of ethanol that decreased 1,8-ANS binding by 50%) of 658 mmol. Preincubation of BSA with ethanol significantly decreased cis-parinaric acid binding to BSA, indicating interaction of ethanol with hydrophobic fatty acid-binding site(s) on BSA. Furthermore, ethanol was found to act on three of the five fatty acid-binding sites on BSA. These data indicated selectivity in the interaction of ethanol with hydrophobic sites on BSA. 13C NMR multiplet relaxation was used to characterize the interaction of ethanol with binding sites on BSA. Detailed analysis of [13C]ethanol relaxation data obtained in the presence of increasing BSA concentrations (25-200 mg/mL) led to the conclusion that the ethanol methyl group, as opposed to its hydroxyl group, binds in a hydrophobic pocket(s) on the protein. Ethanol-induced changes in activity of certain proteins may result from direct binding of ethanol to specific hydrophobic binding sites and/or displacement of endogenous ligands from those sites.