The activity of the TGF-α-like ligand Spitz in Drosophila depends on Rhomboid, a seven-transmembrane spanning protein that resides in the Golgi and acts as a serine protease to cleave Spitz, thereby releasing the soluble ligand. Several rhomboids in Drosophila have been implicated in the processing of TGF-α-like ligands, and consequent EGF receptor activation. The larger number of TGF-α-like ligands in vertebrates raises the possibility that they too might be subject to regulation by rhomboid-like proteins. We present the cDNA cloning and polypeptide sequence of an atypically long human rhomboid, which, based on the absence of critical residues for serine protease activity, is not predicted to act as a serine protease. We examined its tissue distribution, in comparison with TGF-α and the TGF-α-related protein HB-EGF, and the EGF/TGF-α receptor, in mouse embryo. This rhomboid, named p100hRho or RHBDF1, is a seven-transmembrane protein with a long N-terminal cytoplasmic extension that comprises half of the polypeptide sequence, and is found in the endoplasmic reticulum and Golgi, but not on the cell surface. It is expressed as two forms with different lengths, forms dimers and interacts with TGF-α ligands through a luminal interaction with the EGF core ectodomain. Finally, we evaluated the function of p100hRho/RHBDF1 in Drosophila, demonstrating that the short, but not the full-length form has functional activity. The characterization of this protein extends our understanding of the rhomboid family of regulatory proteins. Developmental Dynamics 233:1315–1331, 2005. © 2005 Wiley-Liss, Inc.