Tie1 is an endothelial receptor tyrosine kinase essential for development and maintenance of the vascular system. Here we report generation of transgenic mice expressing enhanced green fluorescent protein (EGFP) or a chimeric protein consisting of a Zeosin resistance marker and EGFP under the control of mouse Tie1 promoter. Intravital monitoring of fluorescence showed that the EGFP reporter recapitulates the Tie1 expression pattern in the developing vasculature, and flow cytometry using EGFP allowed the isolation of essentially pure Tie1-expressing endothelial cells from transgenic mouse embryos. However, EGFP and LacZ transgenic markers were strongly down-regulated in the adult vasculature; unlike the Tie1-LacZ knock-in locus, the promoter was not reactivated during tumor neovascularization, indicating the presence of additional regulatory elements in the Tie1 locus. Starting at midgestation, Tie1 promoter activity became stronger in the arterial than in the venous endothelium; in adult mice, promoter activity was observed in arterioles, capillaries, and lymphatic vessels, indicating a significant degree of specificity in different types of endothelial cells. Our results establish Tie1-Z/EGFP transgenic mice as a useful model to study embryonic vascular development and a convenient source for the isolation of primary endothelial cells.