A negative, regulatory DNA element from the mouse cellular retinoic acid-binding protein I gene promoter was identified. This DNA element, located approximately 1 kilobase upstream from the transcription initiation site of this gene, contained a pair of direct repeats (DRs) separated by 4 base pairs (DR4, TGACCTTTGGGGACCT). By examining a series of reporters deleted or mutated within this DR4 region, it was concluded that the core sequence of this DR4, including both repeats and the spacer, was required for suppressive activity in the mouse embryonal carcinoma cell line P19. From gel retardation experiments, it was concluded that both repeated sequences were essential for specific protein binding, but the spacer sequence was not as critical. Specific residues required for protein binding to this DR4 were identified. In P19 cells, retinoic acid induced the binding of nuclear factors to DR4 and suppressed the activities of the reporters containing this DR4. Co-expression of retinoic acid receptor β or thyroid hormone receptor β1 (T3R(β1)) significantly inhibited the expression of this reporter in P19 cells. Gel retardation with in vitro-synthesized nuclear receptors demonstrated specific binding of this DR4 by T3R(β1) monomers, homodimers, or heterodimers of T3R(β1)/retinoid receptor X β. A biological function of DR4 in crabp-I gene regulation in P19 cells was suggested.