Cordyceps militaris has been previously shown to invert the chirality of (R)-2-phenylpropionic acid to its antipode in the absence of any other biotransformation. To investigate the mechanism of this unusual biotransformation, (R,S)-[2-H-2(1)]-2-phenylpropionic acid, (R,S)-[2-H-2,3,3,3-H-2(3)]-2-phenylpropionic acid, and (R,S)-[3,3,3-H-2(3)]-2-phenylpropionic acid were synthesized and incubated with C. militaris. NMR spectroscopy showed that deuterium exchange of the alpha-methine proton occurred during the inversion process but there was no exchange of the beta-methyl protons. There were no significant differences in the rates of chiral inversion of the three deuterated derivatives and the undeuterated compound, and with all the compounds attaining 84% enantiomeric excess of the (S)-enantiomer after 48 h incubation. The deuterium exchange is not therefore the rate limiting process in this biotransformation The recovery profiles demonstrated that there was rapid metabolism of the beta-methyl deuterated derivatives which did not occur for the undeuterated 2-phenylpropionic acid or the derivative which was only deuterated at the alpha-carbon. These studies clearly illustrate that C. militaris is a useful in vitro model of the chiral inversion of 2-arylpropionic acid derivatives in mammalian systems.