A series of studies was conducted to investigate the potential of (R)- and (S)-2-phenylpropionic acid (2-PPA) to undergo enantioselective covalent binding to protein in freshly isolated rat hepatocytes and to determine whether such covalent binding is dependent on acyl glucuronidation or acyl-CoA formation of 2-PPA. Hepatocytes were incubated with (R,S)-, (R)-, or (S)-[1,2-(14)C(2)]-2-PPA (1 mM), and aliquots of the incubation mixture analyzed for covalent binding, acyl glucuronidation, and acyl-CoA formation over a 3 h period. Covalent binding of 2-PPA to hepatocyte protein was shown to be time-dependent and to be 4.5-fold greater for the (R)-isomer than the (S)-isomer after 3 h of incubation. The enantioselectivity of covalent binding correlated with the enantioselectivity of acyl-CoA formation (R/S = 7.0), but not with acyl glucuronidation (R/S = 0.67) of (R)- and (S)-2-PPA isomers during the 3 h incubation. Inhibition experiments were performed with (R,S)-[1,2-(14)C(2)]-2-PPA (1 mM) incubated with hepatocytes in the presence or absence of trimethylacetic acid (2 mM) or (-)-borneol (1 mM) for the inhibition of 2-PPA-CoA formation and 2-PPA acyl glucuronidation, respectively. Covalent binding of 2-PPA to hepatocyte protein exhibited a 53% decrease in cells treated with trimethylacetic acid, where a 66% decrease in 2-PPA-CoA formation occurred. Conversely, treatment with (-)-borneol, which completely inhibited 2-PPA acyl glucuronidation, only decreased covalent binding by 18.7%. These results indicate that metabolism of 2-PPA by acyl-CoA formation leads to the generation of reactive acylating CoA-thioester species that can contribute to protein covalent binding in a manner that is more extensive than the respective acyl glucuronides.