We previously noted that the initial receptor by which murine osteoclast precursors bind matrix is the integrin α(v)β5 and that granulocyte- macrophage colony-stimulating factor (GM-CSF) decreases expression of this heterodimer by suppressing transcription of the β5 gene. We herein report cloning of the β5 integrin gene promoter and identification of a GM-CSF- responsive sequence. A 13-kilobase (kb) genomic fragment containing part of the β5 gene was isolated by screening a mouse genomic library with a probe derived from the most 5'-end of a murine β5 cDNA. A combination of primer extension and S1 nuclease studies identifies two transcriptional start sites, with the major one designated +1. A 1-kb subclone containing sequence -875 to + 110 is transcriptionally active in a murine myeloid cell line. This 1-kb fragment contains consensus binding sequences for basal (Sp1), lineage- specific (PU.1), and regulatable (signal transducer and activator of transcription) transcription factors. Reflecting our earlier findings, promoter activity is repressed in transfected myeloid cells treated with GM- CSF. Using deletion mutants, we localized a 109-base pair (bp) promoter region responsible for GM-CSF-inhibited β5 transcription. We further identified a 19-bp sequence within the 109-bp region that binds GM-CSF- induced nuclear proteins by gel shift/competition assays. Mutation of the 19- bp sequence not only ablates its capacity to bind nuclear proteins from GM- CSF-treated cells, in vitro, but the same mutation, when introduced in the 1- kb promoter, abolishes its ability to respond to GM-CSF treatment. Northern analysis demonstrates that cycloheximide treatment abrogates the capacity of GM-CSF to decrease β5 mRNA levels. In summary, we have identified a 19-bp cis-element mediating GM-CSF-induced down-regulation of β5 by a mechanism requiring protein synthesis.