The hydrolysis and acyl migration of biosynthetic S-naproxen-β-l-O-acyl glucuronide (I) and R-naproxen-β-l-O-acyl glucuronide (II) was followed by HPLC. Nine first-order kinetic rate constants for the hydrolysis and acyl migration between the β-l-O-acyl glucuronide, its α/β-2, α/β-3-, α/β-4-, and α-1-O-acyl isomers and naproxen aglycone were determined for I and II at pH 7.00, 7.40 and 8.00 at 37°C by kinetic simulation. For I the 3-O-acyl isomer was the most stable isomer as the pseudo-equilibrium ratio for the major acyl-migrated isomers was 1:1.5:0.9 (2-O-acyl isomer:3-O-acyl isomer:4-O-acyl isomer). The 3- and 4-O-acyl isomers of II were equally stable as the pseudo-equilibrium ratio for the major acyl-migrated isomers was 1:1.4:1.4 (2-O-acyl isomer:3-O-acyl isomer:4-O-acyl isomer). For both I and II, the pseudo-equilibrium ratio between the major 2-O-acyl isomer and the minor α-l-O-acyl isomer was 10:1 (2-O-acyl isomer:α-l-O-acyl isomer). The pseudo-equilibrium found for the major acyl-migrated isomers of I and II in the present study corresponds with the pattern previously published for R- and S-ketoprofen-β-l-O-acyl glucuronide acyl-migrated isomers, suggesting that these findings may be general for acyl-migrated β-l-O-acyl glucuronides of enantiomeric 2-arylpropionic acids. Chirality 14:305–312, 2002. © 2002 Wiley-Liss, Inc.