The expression of intercellular adhesion molecule 2 (ICAM-2) in adult tissues is restricted to vascular endothelial cells and megakaryocytes. We have previously shown that the endothelial-specific in vivo activity of the human ICAM-2 promoter is contained within a small (0.33-kilobase (kbp)) 5-flanking region of the gene. Here we describe the in vitro characterization of this region. The ICAM-2 promoter is TATA-less, and transcription in endothelial cells initiates at four sites. Reporter gene expression directed by the promoter was 125-fold greater than vector alone in bovine aortic endothelial cells but less than 2-fold vector alone in non-endothelial (COS) cells, confirming that specificity in vivo was paralleled in vitro. The addition of 2.7 kbp of 5-flanking region to the 0.33-kbp fragment had no effect on promoter activity or specificity. The mutation of an Sp1 motif centered at base pair ؊194 or an eight-base pair palindrome at ؊268 each reduced promoter activity by 70%. Mutation of GATA motifs at ؊145 and ؊53 reduced promoter activity by 78 and 61%, respectively. Specific binding of bovine aortic endothelial cells nuclear proteins to the Sp1 and GATA sites was demonstrated by gel shift analysis. Promoter activity in COS cells was transactivated 3-4-fold by overexpression of GATA-2. The results presented here suggest that transcription from the ICAM-2 promoter in endothelial cells is regulated by the interplay of several positive-acting factors and provide the basis for further analysis of endothelial-specific gene expression.