Phenylbutazone (PB) is known to be biotransformed to its O- and C-glucuronide. Recently, we reported that PB C-glucuronide formation is catalyzed by UGT1A9. Interestingly, despite UGT1A8 sharing high amino acid sequence identity with UGT1A9, UGT1A8 had no PB C-glucuronidating activity. In the present study, we constructed eight UGT1A9/UGT1A8 chimeras and evaluated which region is important for PB C-glucuronide formation. All of the chimeras and UGT1A8 and UGT1A9 had 7-hydroxy-(4-trifluoromethyl)coumarin (HFC) O-glucuronidating activity. The K m values for HFC glucuronidation of UGT1A8, UGT1A9 and their chimeras were divided into two types, UGT1A8 type (high K m) and UGT1A9 type (low K m), and these types were determined according to whether their amino acids at positions 69–132 were those of UGT1A8 or UGT1A9. Likewise, PB O-glucuronidating activity was also detected by all of the chimeras, and their K m values were divided into two types. On the contrary, PB C-glucuronidating activity was detected by UGT1A9 (1–132)/1A8 (133–286), UGT1A9 (1–212)/1A8 (213–286), UGT1A8 (1–68)/1A9 (69–286), and UGT1A8 (1–68)/1A9 (69–132)/1A8 (133–286) chimeras. The region 1A9 (69–132) was common among chimeras having PB C-glucuronidating activity. Of interest is that UGT1A9 (1–68)/1A8 (69–132)/1A9 (133–286) had lost PB C-glucuronidation activity, but retained activities of PB and HFC O-glucuronidation. These results strongly suggested that amino acid positions 69–132 of UGT1A9 are responsible for chemoselectivity for PB and affinity to substrates such as PB and HFC.