Optical isomerization of 2-phenylpropionic acid (hydratropic acid, HTA) was studied in the organs of male rat in vitro. (R)-(-)-HTA was not isomerized by rat liver homogenate even after the addition of CoA, ATP, and Mg2+ to the incubation mixture; however, it was isomerized slowly but significantly in liver slices. This suggests that some additional factor(s) are required for the optical isomerization of HTA in rat liver homogenate. Kidney slices showed about 3-fold higher isomerizing activity of (R)-(-)-HTA than liver slices. (S)-(+)-HTA was also slightly isomerized to (R)-(-)-HTA in kidney slices suggesting that the isomerization of HTA is stereoselective but not stereospecific. The liver and the kidney were considered to be the major organs for isomerization of HTA, since the isomerizing activity of the small intestine was low and other tissues examined were inactive. Change of the incubation medium of liver slices from Krebs-Ringer bicarbonate buffer to Krebs-Ringer buffer without bicarbonate, 0.25 M sucrose solution with 0.05 M Tris-HCl buffer (pH 7.4) or 0.9% NaCl solution with 0.05 M Tris-HCl buffer (pH 7.4) increased the isomerization rate of (R)-(-)-HTA to (S)-(+)-HTA, but decreased the glucuronide formation. During incubation of racemic HTA with liver slices, (S)-(+)-enantiomer percentage in HTA acyl glucuronide (HTA-G) increased with time, whereas that of the free HTA remained nearly constant. These results suggested the stereoselective hydrolysis of (R)-(-)-HTA-G in liver slices, which was confirmed by the results from incubation of HTA-G with rat liver preparations. Kidney homogenate also preferentially hydrolyzed (R)-(-)-HTA-G.