The first solution state structural analysis (NMR) of the C-terminal sequence of human GL that binds to glycogen phosphorylase a (GPa), PEWPSYLGYEKLGPYY-NH2 (1), showed it to be in a random coil conformation. This was supported by molecular dynamics simulation (modelled in solution) using NAMD 2.6. The conformational ambiguity of the peptide makes the structural arrangement of the peptide (and internal residues) strongly dependent on the environment. Thirteen tetra-peptide fragments of the C-terminal sequence, YEKLG-NH2, and the corresponding tri- and di-peptide sequences were used in a fragment screen against GPa. Compound 2 (H-GPYY-NH2) did not give an IC50 value, whereas PEWPSYLGYEKLGPYY-NH2 (1) displayed an IC50 of 34 占 against GPa. Truncated peptides derived from 1, (EKL-NH2, EKLG-NH2, and AcEKNH2) inhibited GPa (21%, 32%, 63%, respectively at 22 mM). These studies suggest key residues within the peptide chain have additional molecular interactions with GPa. The interaction of intra-sequence residues in combination with the terminal residues of PEWPSYLGYEKLGPYY with GPa may form the basis for the design of new inhibitors of GPa. Copyright 頲009 European Peptide Society and John Wiley & Sons, Ltd.