An enzyme immunoassay (EIA) for the determination of paeoniflorin (PA), which is a principal constituent of Chinese paeony root ("Shakuyaku"in Japanese), was established by the use of 6'-hemisuccinyl (III) and 6'-hemiglutaryl PA (IV) as haptens. Compounds III and IV were coupled with β-galactosidase (β-Gal) and bovine serum albumin (BSA) by the N-hydroxysuccinimide ester method to give hemisuccinyl PA-β-Gal (IX) (labeled antigen) and hemiglutaryl PA-BSA (X) (immunogen), respectively. A 30000-fold diluted solution of anti-PA antisera elicited in rabbits by immunization with the immunogen was used for the EIA, and the separation of bound and free fractions was performed by the double antibody method using a goat antiserum to rabbit IgG. 7-β-D-Galactopyranosyloxy-4-methylcoumarin was used as the substrate for the fluorometric assay of β-Gal activity. A satisfactory standard curve for EIA of PA was obtained in the range of 1-400 ng/ml and was only slightly interfered with the addition of serum and urine to the assay mixture of EIA. The PA antiserum reacted with oxypaeoniflorin (0.31%) and albiflorin (0.22%) which are also constituents of Chinese Paeony root.