We have isolated molecular clones of genomic mouse DNA spanning 55 kb, including the entire coding region of the murine alpha1(I) collagen (Col1a1) gene and 24 kb of 5' and 13 kb of 3'-flanking sequences, and have performed a detailed chromatin structure analysis of these sequences. Several new DNase-I-hypersensitive sites were identified. The distal 5'-flanking region contains two clusters of DNase-I-hypersensitive sites located between 7 and 8 kb and between 15 and 20 kb upstream of the start site of transcription, respectively. Several of these sites were shown to be present in collagen-producing, but not in non-producing cells, indicating that they are associated with transcription of the gene and may function in its regulation. One strong constitutive DNase-I-hypersensitive site at -18.5 kb was also cleaved by endogenous nucleases. The 3'-flanking region of the gene contains a DNase-I-hypersensitive site located 6 kb downstream of the end of the gene, as well as sequences that can induce a non-B DNA structure. Because these latter sequences coincide with DNase-I-hypersensitive sites in the homologous human gene, our results suggest that some regulatory elements may play a role in gene regulation, not by specific protein-DNA interactions but by virtue of their ability to induce a non-B DNA structure and/or an alternate chromatin conformation. A comparison of the murine and human Col1a1 domains shows a similar, although not identical, distribution of DNase-I-hypersensitive sites, indicating a conserved arrangement of regulatory elements. Our results strongly suggest that these new sites constitute regulatory elements which are involved in the transcriptional regulation and/or chromatin loop organization of the Col1a1 gene, and they are now amenable for functional analyses.