Amphiphilic peptides can form bottom-up-designed self-assembled hydrogels composed of elongated fibril matrices that could find uses in various biologically-related systems, acting as platforms for drug delivery or scaffolds that mimic extracellular matrices in tissue regeneration systems. We have previously reported that the amphiphilic and anionic β-sheet forming peptide, Pro-Asp-(Phe-Asp)5-Pro, PFD-5, generates hydrogels that template calcium-phosphate mineral and as such, were able to enhance bone formation in vivo. Our earlier results prompted us to further exploit the effects of pH and calcium ion concentration on PFD-5 peptide in solution, in hydrogels and in mineral-loaded hydrogel compositions. Circular dichroism-based characterization of solutions of the peptide demonstrated transitions between the unfolded state to a β-sheet structure as function of peptide concentration, pH and calcium ion concentration. FTIR measurements were employed to monitor differences between the structure of the peptide in solution and in hydrogels. Rheology and dissolution studies demonstrated the improved stability of hydrogels prepared by a two-step procedure, where the peptides are dissolved and self-assemble in the first step, while in the second step, calcium ions are allowed to adsorb onto the system. These results, highlighting the effects of a few central factors on the structure, assembly and stability of amphiphilic and anionic β-sheet peptide systems, will contribute to the further development of designed self-assembled peptide systems from solutions to hydrogels and hydrogel-loaded matrices, such as mineral putty compositions. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 760–772, 2013.