An 163-bp fragment of the rat cytochrome P450 gene, CYP2B2 has been shown to contain sequences that mediate phenobarbital (PB) responsiveness of this gene. In studies on this rat gene and the orthologous mouse gene, Cyp2b10, the minimal fragment required for near full PB responsiveness has varied from about 50 to 80 bp depending on the gene used and the number of copies of the PB responsive sequences assessed. Since there is a single copy of the CYP genes in the genome, we have evaluated deletion and block mutations across an 84-bp region of the PB responsive unit (PBRU), by in situ transfection in rat liver using single copies of the PBRU sequences. From the 5' end, deletions to -2243 retained more than 50% responsiveness to PB compared to the 163-bp fragment. The fragment -2237 to -2155 retained less than 20% responsiveness even though it contained the nuclear receptor (NR)-1, NR-2, and NF-1 motifs which are present in the core of the PBRU. From the 3' end, deletions from -2170 to -2194 eliminated PB responsiveness indicating that the 74-bp sequence from -2243 to -2170 is able to mediate full PB responsiveness. Block mutations within the NR-1 and NF-1 regions reduced responsiveness most dramatically, but did not abolish it, and mutations 3' of the NF-1 site modestly reduced responsiveness. Protein binding was not affected by mutations in the NR-1 region as assessed by DNase I footprinting in vitro but mutations within the NR-2 region reduced binding to the NF-1 site. Mutations of the 5' half or the 3' half of the bipartite NF-1 site, resulted in loss of protection of the NF-1 site and new footprints to the 3' or 5' side, respectively, of the NF-1 site. These results indicate that sequences in addition to the NR-1 and -2 and the NF-1 sites are required for full responsiveness to PB and suggest that proteins which bind to these sites may interact.