The role of chromatin organization in hormone action has been addressed utilizing chimeras between the MMTV-LTR and the bovine papilloma virus (BPV) 69% transforming fragment. These chimeras replicate uniquely as episomal elements in murine fibroblasts and maintain stable high-copy extrachromosomal copy numbers. It was found that nucleosomes are non-randomly organized on the sequences immediately upstream from the MMTV cap site and that this phased structure is independent of hormone induction. A DNase I hypersensitive site is introduced into the chromatin structure upon hormone induction; the location of this site correlates precisely with the sequences required for transfer of hormone regulation in a biological assay. Thus a highly organized nucleoprotein structure serves as a template for hormone regulation in vivo. The episomal elements can be extremely purified from cellular chromatin by selective extraction from the nuclear fraction, sedimentation through sucrose gradients, and concentration. The purified minichromosomes retain their association with 2 to 6 moles of glucocorticoid receptor subunit; this association is specific in that no bound receptor is found with control episomes not containing the MMTV LTR sequences. The fraction is highly enriched, with Greater than 90% of the DNA in the fraction of episomal original. These mini chromosomes should serve as useful templates for in vitro transcription studies.