Liver-selective transcription of the gene for rat arginase, an ornithine cycle (urea cycle) enzyme, is induced by glucocorticoids in a delayed secondary manner; the mRNA induction by the hormones requires de novo protein synthesis, and is preceded by a time lag of several hours. We searched for a DNA element mediating the glucocorticoid induction of the arginase gene with a transient transfection system using hepatoma cell lines. Within the 233-base pair region that is located 11 kilobases downstream from the transcription start site and that spans the junction of intron 7 and exon 8, we detected an enhancer element that is glucocorticoid-responsive and hepatoma cell-selective. The time course of the glucocorticoid induction through this enhancer element was delayed compared to that through the primary glucocorticoid-responsive mouse mammary tumor virus promoter. Footprint analysis revealed four protein-binding sites in this enhancer region. In gel retardation analysis, each site exhibited a complicated profile characterized by a number of shifted bands, some of which were tissue-selective and others ubiquitous. Gel shift competition and antibody supershift/inhibition analysis demonstrated that two of the four sites are recognized by members of the CCAAT/enhancer binding protein (C/EBP) family, some of which are liver-enriched.