Synthesis of avian apo very-low-density lipoprotein (apoVLDL)II is estrogen dependent and liver specific. Competence to express the apoVLDLII gene is not acquired until days 7-9 of embryogenesis and thus lags 5-6 days behind appearance of the liver primordial bud. It is not known whether the delayed ability to activate the gene is attributable to hepatic estrogen receptor profiles, or a requirement for other transcription factors not expressed at earlier stages of embryogenesis. The latter possibility is supported by developmental alterations in nuclease hypersensitivity flanking the gene that occur independently of estrogen administration. We have examined the influence of these hypersensitive regions on expression from the apoVLDLII promoter and have characterized novel protein-DNA interactions at two of them. One is located in a copy of the CR1 family of middle repetitive elements approximately 3.0 kb upstream from the start of the gene. We demonstrate by DNase I footprinting that the site contains an element which matches a predicted consensus silencer sequence. The other site contains no previously identified binding motifs. It is located between nucleotides -228 and -245 and is adjacent to an imperfect estrogen response element (ERE) that we demonstrate acts additively with a canonical ERE 30 nucleotides downstream. We have identified ubiquitous and liver-specific factors that display overlapping DNA contacts with the site. Mutation of G residues contacted by these proteins decreases hormone-inducible expression from the promoter 5- to 8-fold. Hepatic levels of the liver-enriched factor interacting with this site increase abruptly between days 7 and 9 of embryogenesis, suggesting that it may be an important determinant of the ability to express the apoVLDLII and possibly other liver-specific genes.