In an attempt to identify potential folding initiation sites for a small, all beta-protein domain, we have examined the conformational preferences in aqueous solution of peptides that span the entire length of the alpha-spectrin SH3 domain, using proton nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. Two of the peptides correspond to beta-hairpins (m6 and m8), one to the RT-loop (m4, which can be considered as a distorted beta-hairpin), one to a beta-hairpin created by joining the N and C-terminal strands via a small linker (m2) and the fifth one to a three-stranded antiparallel beta-sheet composed of beta-hairpins m6 and m8 (m68). To estimate the distorting effect of the aromatic side-chains of Trp41 and Trp42 on the CD and NMR spectra of peptides m6, m8 and m68, we have also analyzed a short, ten-residue random-coil peptide containing residues 39 to 44 (mC). The CD and NMR results indicate that none of the peptides populates to a large extent a particular secondary structure conformation. However, careful anlaysis of the NMR data reveals that peptides m6, m8 and m68 could adopt, to a small extent, native-like conformations, although in the case of peptide m68 there is also evidence of the presence of non-native helical conformations. Addition of 30% (v/v) 2,2,2-trifluoroethanol stabilizes the appearance of non-native helical populations in some small regions of peptides m2, m4, m8 and m68, while it induces a native-like conformation in peptide m6. Those fragments corresponding to the two real beta-hairpins in the protein are the ones which exhibit some tendency to populate native-like structures (m6 and m8), while the ones corresponding to the long RT-loop (m4) or the newly created one (m2) are mainly unstructured in water solution. Although there could be some local interactions that favor the acquisition of a native secondary structure in this domain, tertiary interactions should play a major role in defining its native secondary structure.