The receptor tyrosine kinase Flk-1 plays a pivotal role in the development of the vascular system and in the vascularization of a wide variety of tumors. We have investigated the activity of cis-acting sequences of the murine Flk-1gene in the tumor endothelium of experimental tumor models in vivo. B16 melanoma, BFS-1 fibrosarcoma, and polyoma middle T-induced mammary adenocarcinoma were grown in transgenic mice that express the LacZ reporter gene under the control of a 939-bp Flk-1 promoter fragment and an enhancer element located in a 2.3-kb fragment of the first intron. In all experimental tumor models examined, strong endothelium-specific reporter gene expression was observed while being absent from most blood vessels in normal adult tissue. The expression patterns of the LacZreporter gene correlate well between established tumors grown in Flk1-LacZ transgenic mice and tumors grown in Flk-1 +/LacZ knock-in mice that express the LacZ reporter gene from the endogenous Flk-1 locus. The endothelium-specific activity of the Flk-1 promoter/enhancer sequences in three different experimental tumor models demonstrates that the regulatory sequences that mediate the up-regulation of Flk-1 in the tumor endothelium are contained in the Flk-1promoter/enhancer sequences used, and that these elements function relatively independently of the tumor type. The Flk-1promoter/enhancer sequences should allow the analysis of the signaling pathways that lead to the up-regulation of Flk-1 in the tumor endothelium and to specifically target therapeutic genes to the endothelium of tumors for antiangiogenic tumor therapy.