A distal 163-bp fragment mediates phenobarbital responsiveness of the rat CYP2B2 gene. Multiple cis-acting elements in this fragment cooperate to form a phenobarbital response unit (PBRU). A nuclear factor 1 binding site and an associated nuclear receptor hexamer half-site are present in both the rat CYP2B2 PBRU and the homologous mouse Cyp2b10 sequence. Based on mutational analyses, the hexamer half-site has been reported to act positively in CYP2B2 and negatively in Cyp2b10. However, the specific mutations introduced into the rat and mouse hexamer half-sites were different, raising the possibility that the different roles attributed to the element may be a consequence of the different mutations used. We introduced into the rat CYP2B2 hexamer half-site the specific mutational change previously introduced into the Cyp2b10 sequence, where its effect was to increase the basal level of expression and to abolish phenobarbital responsiveness. In the rat context, this mutation reduced but did not abolish phenobarbital responsiveness and decreased, rather than increased, the basal level of expression. The residual phenobarbital responsiveness of the hexamer half-site mutant, as well as that of nuclear factor 1 mutants, indicates that these elements behave as positive accessory sites, suggesting that factors binding to them function as activators of phenobarbital-dependent transcription.