Mitochondrial coupling factor-6 (CF-6) is a component of the ATP synthase complex essential for energy transduction. CF-6, which is localized to the surface of endothelial cells (ECs) and released by shear stress, has been implicated as an endogenous vasoconstrictor. Previous methods of obtaining CF-6 through purification and recombinant methods were laborious and inefficient. Here, we describe the chemical synthesis of human CF-6, (33-108)-NH2, its C-terminal fragment (55-108)-NH2, which is termed pCF-6; the rat CF-6, (33-108)-NH2, its C-terminal fragment pCF-6, (55-108)-NH2; and two N-terminal fragments of the rat pro-coupling factor-6, (24-52)-NH2 and (33-52)-NH2. Biological activities of each peptide were initially screened with bioassays and verified by in vivo studies. Accordingly, intravenous administration of CF-6, pCF-6, rat CF-6, and rat pCF-6 produced a modest but statistically significant increase in blood pressure and heart rate in urethane anesthetized rats, whereas the N-terminal rat pro-coupling factor-6, (24-52)-NH2 and (33-52)-NH2 caused no significant pressor response. Thus, the biologically active site probably resides at the C-terminal portion of CF-6 peptides.