A 15 amino acid long selenopeptide (15SeP) was recently reported to possess nearly the same catalytic activity as glutathione peroxidase for the reduction of hydrogen peroxide by glutathione [Sun, Y. et al. (2004) J. Biol. Chem. 279, 37235-37240]. Such a finding is startling considering the high efficiency of the natural enzyme and the modest catalytic properties of most short peptides. As 15SeP had been subjected only to limited chemical characterization, we prepared it by a new route involving selenocysteine-mediated native chemical ligation. High resolution MALDI mass spectrometry confirmed the identity of the reaction product, while circular dichroism spectroscopy showed that 15SeP assumes a random coil conformation in solution. Although low levels of peroxidase activity were detectable under standard assay conditions, the peptide is more than five orders of magnitude less active than native Gpx. Our observations are incompatible with claims ascribing remarkable catalytic properties to 15SeP, and suggest that the efficiency of glutathione peroxidase derives from its well-defined three-dimensional structure.