We previously cloned the human cellular retinoic acid-binding protein-II (CRABPII) gene and demonstrated a rapid and transient increase in retinoic acid (RA)-dependent transcription in cultured human skin fibroblasts. To determine whether retinoid receptors could regulate CRABPII gene transcription, cotransfection experiments were performed. When RAR alpha was cotransfected in Cos-1 cells with a reporter construct containing -8.0 kilobases of the upstream region, an 18-fold RA induction was obtained. By deletion analysis, a region essential for RA induction located approximately -5.6 kilobases upstream from the human CRABPII gene start site was identified. Sequencing and mutational analysis identified a direct repeat (GGGTCAttggaAGGACA) with 5-base pair spacing (DR-5) that is critical for RA-mediated induction of human CRABPII gene transcription. This is different from the mouse CRABPII gene in which two RAREs (DR-1 and DR-2) are required for full activation. To determine whether RAR and RXR can bind to the human CRABPII RARE, gel retardation assays were performed. In these assays, in vitro translated RAR alpha and RXR alpha were found to bind efficiently as heterodimers in gel retardation assays; weak binding of RAR alpha homodimers was observed. These data demonstrate that the human CRABPII gene is regulated by a far upstream RARE that most efficiently binds RAR-RXR heterodimers.