Endothelin-converting enzymes (ECEs) are the key enzymes in the endothelin (ET) biosynthesis that catalyze the conversion of big ET, the biologically inactive precursor of mature ET. Two enzymes, termed ECE-1 and ECE-2, have been molecularly identified. Here, we report novel four subisoforms of ECE-2 that differ in their N-terminal cytoplasmic tails, termed ECE-2a-1, ECE-2a-2, ECE-2b-1, and ECE-2b-2. RT-PCR analysis of these subisoforms in bovine tissues demonstrated that their tissue distribution was strikingly different. ECE-2a-1 and ECE-2a-2 are expressed in a variety of tissues including liver, kidney, adrenal gland, testis, and endothelial cells, while ECE-2b-1 and ECE-2b-2 are expressed abundantly in brain and adrenal gland. Furthermore, ECE-2a-1 and ECE-2b-2 were revealed to be predominant forms as compared to ECE-2a-2 and ECE-2b-1, respectively. Immunohistochemical analyses of CHO cells, stably expressing ECE-2a-1 or ECE-2b-2, revealed that both ECE-2a-1 and ECE-2b-2 were localized in intracellular compartments but not on the cell surface. Detailed analysis of ECE-2 subisoforms will provide crucial information to clarify the physiological function of ECE-2.