Understanding the self-assembly of peptides into ordered nanostructures is recently getting much attention since it can provide an alternative route for fabricating novel bio-inspired materials. In order to realize the potential of the peptide-based nanofabrication technology, however, more information is needed regarding the integrity or stability of peptide nanostructures under the process conditions encountered in their applications. In this study, we investigated the stability of self-assembled peptide nanowires (PNWs) and nanotubes (PNTs) against thermal, chemical, proteolytic attacks, and their conformational changes upon heat treatment. PNWs and PNTs were grown by the self-assembly of diphenylalanine (Phe–Phe), a peptide building block, on solid substrates at different chemical atmospheres and temperatures. The incubation of diphenylalanine under aniline vapor at 150°C led to the formation of PNWs, while its incubation with water vapor at 25°C produced PNTs. We analyzed the stability of peptide nanostructures using multiple tools, such as electron microscopy, thermal analysis tools, circular dichroism, and Fourier-transform infrared spectroscopy. Our results show that PNWs are highly stable up to 200°C and remain unchanged when incubated in aqueous solutions (from pH 1 to 14) or in various chemical solvents (from polar to non-polar). In contrast, PNTs started to disintegrate even at 100°C and underwent a conformational change at an elevated temperature. When we further studied their resistance to a proteolytic environment, we discovered that PNWs kept their initial structure while PNTs fully disintegrated. We found that the high stability of PNWs originates from their predominant β-sheet conformation and the conformational change of diphenylalanine nanostructures. Our study suggests that self-assembled PNWs are suitable for future nano-scale applications requiring harsh processing conditions. Biotechnol. Bioeng. 2010; 105: 221–230. © 2009 Wiley Periodicals, Inc.