Membrane fusion is essential for many biological processes. Though there have been many structure and fusion studies of cellular and viral fusion proteins in the last years, their functional mechanism remains elusive. In particular, the structural modes of operation of the transmembrane domains and viral fusion peptides of fusion proteins during membrane fusion have not been elucidated, although work on de novo designed fusogenic peptides suggested that conformational flexibility was necessary. In addition, the use of different and incompatible measurement criteria has made a comparative overview difficult. Here, we report a systematic structural analysis of viral fusion peptides from different fusion protein classes and transmembrane domains of viral and cellular fusion proteins by using circular dichroism spectroscopy. The data that were obtained demonstrate that class I viral fusion peptides show a structural flexibility between helix and irregular secondary structures, whereas fusion peptides of class II viral fusion proteins are characterized by a stable random coil and turn structure. Thus, conformational flexibility does not seem to be a universal criterion for the fusion activity of a fusion peptide. On the contrary, the transmembrane domains of fusion proteins are distinguished by a structural flexibility between helix and sheet structure that is similar to de novo designed unnatural peptides with high fusion activities (M. W. Hofmann et al. PNAS 2004, 101, 14 776-14 781). Thus, the conformational behavior of the fusogenic unnatural peptides most closely resembles that of fusion protein transmembrane domains, and allows them to be used to gain a deeper understanding of the membrane fusion process. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.