The mouse phenobarbital (PB)-inducible Cyp2b10 gene promoter has been isolated and sequenced, and control of its expression has been characterized. The 1405-base pair (bp) Cyp2b10 promoter sequence is 83% identical to the corresponding region from the rat CYP2B2 gene. In addition to the lack of CA repeats, differences include insertion of 42 base pairs (−123/-82 bp) into the middle of a consensus sequence to the so-called “Barbie box.” In this report, we have developed a primary mouse hepatocyte culture system in which endogenous 2B10 mRNA as well as Cyp2b10-driven CAT activity were induced by PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), but not by the 3-chloro derivative of TCPOBOP. Deletion analysis of the Cyp2b10 promoter identified a basal transcription element at −64/-34 bp and a negative element at −971/-775 bp. Sequences contained within the −1404/-971 bp region are responsible for the induced CAT activity. DNase I protection and gel shift assays detected five major protein binding sites within the −1404/-971 bp fragment, one of which shared high sequence identity with a portion of a regulatory element in CYP2B2 gene (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A.(1995) Gene 158, 263-268). Our results indicate that sequences important for PB-induced transcription of Cyp2b10 gene are located in the distal promoter.