The HMG-box transcription factor Sox17 has been shown to play important roles in both endoderm formation and cardiovascular development. To conditionally inactivate genes in these domains, we have targeted a codon improved Cre Recombinase (iCre) into exon 1 of the Sox17 gene. Surprisingly, Cre-mediated recombination in the Rosa26 reporter mouse line revealed largely specific activity within the vasculature rather than in endoderm-derived tissues. Here we report a new Cre knock-in mouse line, Sox17iCre with activity in the vascular endothelial cells of arteries in the cardiovascular system but not in veins. Cre-mediated recombination was also strongly detected in the liver and spleen, the two organs associated with hematopoiesis. Thus, these results indicate that the Sox17iCre would be an appropriate tool for conditional mutagenesis of genes in the vasculature and could be used in studies of blood vessel development and angiogenesis. Additionally, we provide evidence that two different promoters drive Sox17 expression in the endodermal and vascular system. genesis 47:476–483, 2009. © 2009 Wiley-Liss, Inc.