Human integrin alphavbeta receptors are expressed in a number of cells and their expression is regulated at the level of transcription and by post-transcriptional mechanisms. A substantial body of research exists on the structure, function, molecular biology and physiological significance of alphav integrin receptors. However, the importance of particular cis-acting DNA elements or trans-acting nuclear factors in the regulation of the alphav gene promoter is still not adequately understood. Previous functional analysis of the alphav gene 5' flanking region in transfected cultured cells identified cis elements critical for alphav transcription within a 222-bp region. To define further the location of this enhancing element, we performed DNase I footprinting of the human alphav gene promoter between -522 and the translation initiation site. For this purpose, nuclear extracts of alphavbeta3-positive cells, human umbilical vein endothelial cells, were used. Nuclear proteins of endothelial cells strongly protected essentially one region corresponding to the sequence between -194 and -172 of the alphav promoter region. Electrophoretic mobility shift assays with different oligonucleotides, and competition analysis identified a CTCCTCCTC sequence that is directly involved in the transcriptional activity of the alphav promoter. Purified Sp1 alone produced an identical footprint, and DNA binding assays using anti-Sp1 and anti-Sp3 antibodies showed that transcription factors Sp1 and Sp3 were the major nuclear proteins bound to this region.