BACKGROUND: Targeting protective gene expression to porcine endothelium has obvious advantages in xenotransplantation, but no endothelial cell-specific promoters have yet been used successfully in transgenic pigs. We have previously reported that a human intercellular adhesion molecule-2 (ICAM-2) gene promoter fragment functioned efficiently in transgenic mice but not pigs, suggesting that it lacked important transcriptional signals. In this study, we cloned and characterized regulatory elements of the pig ICAM-2 gene. METHODS: Various segments of the pig ICAM-2 gene upstream region and first intron were cloned into a luciferase reporter vector and assayed for promoter activity in vitro. Putative regulatory elements were analysed by site-directed mutagenesis. RESULTS: A 0.90-kb pig ICAM-2 promoter fragment had strong activity in pig endothelial cells but not in non-endothelial cells. Deletion analysis revealed that the majority of promoter activity was specified by a 0.48-kb sub-fragment with significant homology to the human ICAM-2 promoter. Conserved positive-acting elements included binding sites for GATA and Ets transcription factors, and a palindromic octamer (P(8)) that has been implicated in the endothelial specificity of several genes. Significant enhancer activity was identified within the first intron of the pig ICAM-2 gene. Mutational analysis was used to show that a second P(8) site in the first intron was essential for enhancer activity. CONCLUSIONS: The pig and human ICAM-2 promoters exhibit many similarities, but the pig ICAM-2 gene, unlike its human and mouse homologs, contains P(8) sites in both the promoter and first intron. The enhancer activity associated with the intronic P(8) site suggests that it may be the key to achieving strong endothelial cell-specific transgene expression in pigs.