Core histone octamers reconstituted in vitro onto DNA fragments containing the chicken β-globin gene promoter are precisely positioned with respect to the underlying DNA sequence [1]. Here we show that this is also true of the chicken β-globin gene enhancer. These nucleosome binding sites are also employed within transfected COS cell nuclei, where the chicken β-globin gene is transcriptionally inactive. Similar results were found in vivo, where positioned nucleosomes were detected over the inactive β-globin promoter in chicken brain cells and 5-day red blood cells, and over the inactive β-globin enhancer in brain cells. In contrast, the promoter and enhancer regions were found to be nucleosome-free in 15-day erythrocytes where the β-globln gene Is active. We argue that these results suggest a role for positioned nucleosomes in the regulation of the transcription of the chicken β-globin gene.