The closely linked, reciprocally imprinted mouse genes, insulin-like growth factor II (Igf-2) and H19, provide a significant paradigm for studies of the mechanism of parental imprinting. Most studies have focused on regions within and proximate to the genes, but an analysis of the whole region is essential to unravel how expression of these genes is controlled. A comparative long-range analysis of a 130-kb genomic region containing both genes was therefore carried out using material from normal and chromosome 7 maternal uniparental disomic embryos. We examined DNA methylation, chromatin structure assessed by hypersensitivity to DNase I, and regions that show strong conservation among mammalian species. The critical boundaries of differential DNA methylation and DNase I hypersensitivity are apparently confined within and proximate to the Igf-2 and H19 genes. However, we have identified a novel intergenic region that is conserved in mammals, GC-rich, and unmethylated in embryos and contains major DNase I hypersensitive sites.