DNase I hypersensitive (DH) sites in a 12-kb genomic fragment carrying the mouse EndoA gene were examined to obtain information on the changes in chromatin structure associated with activation of this gene encoding extra-endodermal cytoskeletal protein A (EndoA) during early mouse embryogenesis. Seven DH sites were found in this locus in parietal yolk-sac-like cells, PYS-2, which produce EndoA constitutively. In differentiated mouse teratocarcinoma F9 cells that produce EndoA inductively, this locus has three DH sites. In both cell lines, these sites were mapped to the upstream region of the promoter, the promoter and the 3' enhancer region. The DNA of PYS-2 cells has one more DH site within the first exon and three additional DH sites within the first intron. These DH sites are not present in DNA from BALB/c 3T3 cells and undifferentiated F9 cells that do not produce EndoA. Thus, the formation of these differentiation-dependent DH sites is required for the differentiation-specific expression of the mouse EndoA. In addition, another strong DH site, which may be associated with the B2 element expression, was detected in the third intron of the gene in undifferentiated F9 cells.