The promoter and its upstream regulatory region of the mouse cellular retinoic acid-binding protein I (crabp-I) gene were examined in transgenic mouse embryos, a mouse embryonal carcinoma cell line P19, and a mouse embryonic fibroblast cell line 3T6. In transgenic mouse embryos, a β- galactosidase reporter gene under the control of crabp-I promoter and its upstream regulatory region displayed a very specific pattern of expression characteristic of crabp-I gene expression during developmental stages. In tissue culture systems, the minimal promoter of this gene was identified, and regions containing positive and negative regulatory activities were dissected from the upstream 3-kilobase sequence using assays for transient reporter activity. It is concluded that the minimal promoter of the mouse crabp-I gene is located between 120 and 150 base pairs upstream from the transcription initiation site. Several cell type-specific positive and negative regulatory regions for this promoter have been identified. A region encoding a common negative regulatory activity in both P19 and 3T6 cells is also inhibitory to two heterologous promoters, and specific protein-DNA interactions between this DNA fragment and nuclear extracts of P19 and 3T6 are demonstrated by gel retardation experiments.