Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 gene (Igf2) and the closely linked H19 gene located on distal chromosome 7 is regulated by a 2.4-kb imprinting control region (ICR) located upstream of the H19 gene. In somatic cells, the maternally and paternally derived ICRs are hypo- and hypermethylated, respectively, with the former binding the insulator protein CCCTC-binding factor (CTCF) and acting to block access of enhancers to the Igf2 promoter. Here we report on a detailed in vivo footprinting analysis-using ligation-mediated PCR combined with in vivo dimethyl sulfate, DNase I, or UV treatment-of ICR sequences located outside of the CTCF binding domains. In mouse primary embryo fibroblasts carrying only maternal or paternal copies of distal chromosome 7, we have identified five prominent footprints specific to the maternal ICR. Each of the five footprinted areas contains at least two nuclear hormone receptor hexad binding sites arranged with irregular spacing. When combined with fibroblast nuclear extracts, these sequences interact with complexes containing retinoic X receptor alpha and estrogen receptor beta. More significantly, the footprint sequences bind nuclear hormone receptor complexes in male, but not female, germ cell extracts purified from fetuses at a developmental stage corresponding to the time of establishment of differential ICR methylation. These data are consistent with the possibility that nuclear hormone receptor complexes participate in the establishment of differential ICR methylation imprinting in the germ line.