We have identified four DNAase I-hypersensitive regions (DHRs) at the junB locus. DHR1 is located between sequences -100 and +250, DHR2 is centered at -1000, DHR3 at -1650, and DHR4 at +2040 relative to the junB transcriptional start site. Sequence analysis of these DHRs revealed two serum response elements at -1452 and +2091, two cyclic AMP response elements at +2071 and +2116, and a 12-O-tetradecanoylphorbol-13-acetate (TPA) response element at -949. To study the contribution made by these cis-elements to junB transcriptional regulation, we stably transfected a recombinant mouse junB gene (JBSV4) containing the intact junB coding sequences, 6.3 kb of 5'-flanking DNA, and 2.0 kb of 3'-flanking DNA into Rat1A cells. The pattern of DHRs identified at the mouse junB locus was re-established at the JBSV4 locus. By directly comparing JBSV4 and rat junB mRNA levels, we found that these genes were induced to equivalent levels by serum, TPA, cyclic AMP, platelet-derived growth factor, epidermal growth factor, and basic fibroblastic growth factor. These results established that JBSV4 resides in a physical environment within chromatin that closely mimics that of the junB locus, and contains the necessary sequence information to recapitulate the transcriptional regulation of junB. By analysing a series of recombinant mouse junB genes containing deletion mutations in 5'-flanking and 3'-flanking sequences, we provide a quantitative assessment of the contribution these sequences make to junB induction by different regulatory agents.