The int-3 oncogene was identified as a frequent target in Mouse Mammary Tumor Virus (MMTV)-induced mammary carcinomas and encodes the intracellular domain of a novel mouse Notch gene. To investigate the role of the int-3 proto-oncogene in mouse development and carcinogenesis, we isolated cDNA clones corresponding to the entire coding potential of the int-3 proto-oncogene. We propose to name this gene Notch4 and reserve the int-3 nomenclature for references to the oncogenic form. The deduced amino acid sequence of Notch4 contains conserved motifs found in Notch proteins; however Notch4 has fewer epidermal growth factor (EGF)-like repeats and a shorter intracellular domain than other mouse Notch homologues. Comparison of the coding potential of the int-3 gene to that of Notch4 suggests that loss of the extracellular domain of Notch4 leads to constitutive activation of this murine Notch protein. In situ hybridization revealed that Notch4 transcripts are primarily restricted to endothelial cells in embryonic and adult life. Truncated Notch4 transcripts were detected in post-meiotic male germ cells. The distinct Notch4 protein features and its restricted expression pattern suggests a specific role for Notch4 during development of vertebrate endothelium.