We have isolated recombinant genomic clones encompassing several kilobase pairs of the 5'-flanking regions of both the human and murine gas-1 gene (growth arrest-specific gene 1). Both species share a highly conserved region of approximately 550 base pairs upstream of the gas-1 transcription start site. Deletion analysis of the murine gas-1 promoter demonstrated that a fragment containing the first 665 base pairs is sufficient to drive the serum-regulated expression of a luciferase reporter gene in NIH3T3 cells, in a manner qualitatively reflecting the activity of the endogenous gene. Gel retardation assays indicated the presence of a number of DNA-binding proteins specific for sequences contained within the gas-1 transcription regulatory region. Comparative studies with extracts prepared from growing and resting cells revealed several growth state-specific binding activities. One promoter fragment that bound prominent growth- and arrest-specific complexes was further analyzed by copper-phenanthroline footprinting. It was found that the same DNA element is a target both for growth- and for arrest-specific activities. The factors characterized in this study are the first candidates for transcriptional regulators mediating cell growth-specific repression and/or growth arrest-specific activation of gene expression.