A DNA segment close to the 5' end of the chicken adult beta-globin gene contains a hypersensitive site for nuclease action. Using a variety of endonucleases, we show that this hypersensitive site is in fact an accessible region, which extends from approximately 60 to approximately 260 base pairs 5' from the start of mRNA transcription. The presence of this hypersensitive region is correlated with gene activity; it is observed in nuclei isolated from the definitive erythrocytes of 14-day chicken embryos, but is not observed in nuclei isolated from the primitive erythrocytes of 5-day embryos (which do not express this gene) or in brain nuclei or purified DNA. The close spacing of accessible digestion sites on this DNA segment is not consistent with the presence of a normal nucleosome. Furthermore, a 115 base pair DNA fragment contained within this nuclease-sensitive region can be excised by Msp I digestion and released from nuclei, in at least 50% yield. Approximately one third of the released fragments behave as protein-free DNA.