We have cloned the promoter of the avian beta 3 integrin gene. Using a probe comprising the 5'-untranslated region of an avian macrophage beta 3 cDNA, characterized by 5' rapid amplification of cDNA ends, several clones were isolated from an avian genomic library. One major and one minor transcriptional start site were identified at +1 and -47 base pairs, respectively, with the latter coinciding with a consensus sequence of an initiator. DNA sequence analysis of 800 base pairs 5' of the transcriptional start site fails to reveal either a TATA or CAAT box. In addition to an initiator, the first 200 base pairs contain consensus sequences for the binding of AP-1 and SP-1. A 3.5-kilobase fragment located immediately upstream of the transcriptional start site exhibits functional promoter activity, and deletion analysis reveals both suppressor and enhancer elements. In light of our observation that 1,25-dihydroxyvitamin D3 (D3) accelerates beta 3 transcription, we determined whether the avian beta 3 promoter contains a vitamin D response element (VDRE). Transfected reporter constructs containing the first 1.5 kilobases upstream of the major beta 3 transcriptional start site respond to D3 with enhanced luciferase activity. Analysis of this region reveals a classical VDRE consensus sequence, located at -756 to -770. The following observations support the hypothesis that this sequence represents a functional VDRE: 1) a 600-base pair genomic fragment or a 29-base pair oligomer, each containing the putative VDRE, respond to D3 when transfected into HD11 cells; 2) a 67-base pair DNA fragment derived from genomic DNA and containing the candidate beta 3 VDRE specifically binds the vitamin D receptor-retinoid X receptor beta complex; and 3) avian osteoclast precursor-derived nuclear extracts bind to a synthetic oligomer containing the beta 3 VDRE-like sequence and, in turn, are specifically displaced by unlabeled beta 3 VDRE and anti-vitamin D receptor antibody.