The recently isolated trichogin GA IV is a 10 amino acid, Aib-rich peptide with potent membrane-modifying properties. The peptide is too short to span lipid bilayers, so the mechanism by which trichogin GA IV interacts with biological membranes is unknown. The crystal structure has been solved, but there is much less information on the peptide's conformation in solution. This problem is addressed by examining the electron spin resonance (ESR) of single and double TOAC-labeled trichogin GA IV analogues, where TOAC is a rigid nitroxide amino acid and serves as an Aib analogue. The doubly labeled peptides, trich-1,4, -4,8 and -1,8, represent all possible trichogin GA IV analogues containing two Aib --> TOAC substitutions. ESR in MeOH at 200 K of the g approximate to 2 spectral region suggests that the N-terminus from residues one through four adopts a helical structure similar to that observed in the crystal. However, the central and C-terminal regions appear to be structurally heterogeneous. To further resolve the solution structure, we performed half-field ESR measurements in a MeOH/EtOH glass at 120 K and referenced them against similar measurements from a series of double TOAC-labeled peptides of known structure. Half-field intensities depend on electron spin dipolar coupling and scale as 1/r(6) where r is the internitroxide distance. The combination of allowed (g approximate to 2) and half-field ESR indicates that the trichogin GA IV C-terminal region is partially alpha-helical, as in the crystal structure, but is in equilibrium with unfolded conformers. It is suggested that the Gly-Gly stretch creates a hinge point between two short but stable helical regions. The combined ESR methods used here represent a new approach for determining the solution structures of partially folded peptides.