Pulsed electron-electron double-resonance (PELDOR) and CW-ESR spectroscopies were used to study the dipole-dipole interaction of spin labels in frozen glassy solutions of mono- and double-labeled analogues of the 22-residue, the head-to-tail covalent dimer of the peptaibol antibiotic trichogin GA IV. The [TOAC-1] and [TOAC-1,19] dimers were studied in methanol and in a chloroform-toluene mixture. It was shown these dimer molecules do not form aggregates in methanol. However, in chloroform-toluene, the PELDOR signal of the mono-labeled dimer shows oscillations, due to the dipole-dipole interaction of the nitroxide spin labels. From the signal decay, the number of molecules per aggregate was estimated to be similar to2-3, depending on the concentration used. The contributions of inter- and intra-aggregate interactions of spin labels to the PELDOR signal decay were separated. The distance distribution functions of the intra-aggregate interactions range from 3.0 to 4.5 nm. The maxima are observed at 3.4 nm. From the PELDOR data, we obtained the functions of distance distributions of labels for the monomeric double-labeled peptide in methanol as well as for the same peptide isolated within aggregates of nonlabeled peptide molecules in chloroform-toluene. In the latter case, the distribution function is the most narrow with a maximum at a distance between the nitroxide spin labels of 2.8 nm, which is in agreement with the theoretical distance between labels at corresponding residue positions of an alpha-helical structure.