Native platelet factor 4 (PF4) (70 residues) has a hydrophobic three-stranded anti-parallel P-sheet domain on to which is folded an amphipathic C-terminal α-helix and an aperiodic N-terminal domain. The 33-amino acid β-sheet domain from PF4 (residues 23-55) has been synthesized and studied by c.d. and n.m.r. At 10°C and low concentration, peptide 23-55 appears to exist in aqueous solution in a random-coil distribution of highly flexible conformational states. Some preferred conformation, however, is observed, particularly within a relatively stable chain reversal from Leu-45 to Arg-49. As the peptide concentration and/or temperature is increased, a new conformational state(s) appears and intensifies as slowly exchanging (600MHz 1H-n.m.r. chemical-shift time scale) random-coil resonances disappear. Hill plots of the concentration-dependence indicated mostly tetramer formation as found in native PF4. Although apparent resonance linewidths in aggregate state(s) are of the order of 100 Hz, sequence-specific assignments for most resonances could be made. N.m.r./nuclear Overhauser effect structural analysis indicates the formation of multiple native-like anti-parallel β-sheet conformations, kinetically trapped via sub unit-association-induced hydrophobic collapse and stabilized by low-dielectric electrostatic interactions among/between Gly-28 and Lys-50 in opposing subunits. Results are discussed in terms of protein folding.