The higher ordered structure of the chicken alpha 2(1) procollagen gene was analyzed in chromatin isolated from expressing (lung) and nonexpressing (reticulocyte and erythrocyte) tissues. Digestion of DNA with methylation sensitive restriction endonucleases revealed that this gene was methylated in all tissues examined and that no differences existed in the promoter methylation patterns between expressing and nonexpressing tissues. DNAse 1 hypersensitive sites were located between 100-300 bp upstream from the transcription initiation site and within the first intron. These sites were also hypersensitive to the single-strand specific S1 nuclease, implying that this region of the gene in the chromatin is either in an unfolded single-stranded conformation or under severe conformational stress. These differences in the alpha 2(1) chromatin structure were confirmed by the finding that the promoter was more accessible to restriction endonuclease digestion in the expressing tissues than in the nonexpressing tissues. Digestion of chromatin with Pst I and Sma I revealed that some of these sites in the promoter were differentially protected by DNA-binding proteins in the two tissue types. These protected sites were located as far upstream as -1,600 and downstream within the first intron at +800.