The human integrin beta3 participates in a wide range of adhesive biologic functions and is expressed in a selected subset of tissues, but little is known about the cis-acting DNA elements or trans-acting factors responsible for this regulation. Using cell lines characterized for beta3 expression, a number of upstream regulatory regions in the beta3 gene were identified. (1) The three regions from -1159 to -584, -290 to -146, and -126 to -115 demonstrated positive, negative, and negative activity, respectively. (2) The region from -115 to +29 of the beta3 gene was sufficient for cell-specific activity. Deletion of the sequence from -115 to -89 produced a 6- to 40-fold reduction in reporter gene activity in beta3-expressing megakaryocytic cell lines (K562, Dami, and HEL), but only a 1.7- and 2.7-fold reduction, respectively, in beta3-expressing endothelial and melanoma cell lines, and 1.3- and 2. 8-fold reduction, respectively, in non-beta3-expressing Chinese hamster ovary and 293 cell lines. This sequence also bound nuclear proteins in a cell-specific manner in electrophoretic mobility shift assays. Mutational analysis indicated that the sequence GAGGGG (positions -113 to -108) is a megakaryocytic cell line-specific cis-acting element. (3) The region from -89 to +29 promoted lower activity in all cell lines. We also provide evidence that a CCCACCC sequence at position -70 has transcriptional activity, most likely through the Sp1 transcription factor. These data supply the first detailed map of the transcriptional regulatory elements of the 5' region of the beta3 gene, define positive regulatory sequences with potent megakaryocyte preferential activity, and indicate that the ubiquitous transcription factor, Sp1, may augment beta3 gene expression.