AbstractLow‐energy collision‐induced electrospray ionization tandem mass spectrometry ESI‐CID‐MS/MS (in the positive ion mode) was used for the structural characterization of a series of five representative epioplythiodioxopipreazines: dethiotetra(methylthio)chemotin, chaetocochins A, B and C, and chemotin isolated from the fungus Chaetomium cochliodes. The fragmentation pathways were elucidated by ESI‐IT‐MSn. The elemental compositions of most of the product ions were confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The loss of the S2 molecule seems always to be the first when the SS bond is present. The loss of 77 Da corresponding to the loss of the [CH3SCH2O]' radical was diagnostic for chaetocochins A and B, in which the two piperazines rings are linked by an acetal group. It was found that a McLafferty rearrangement plays a significant role in the skeleton fragmentation of theses series of studied complex multicyclic piperazine compounds. This MacLafferty rearrangement affords the product ions at m/z 416 and 400, containing the two piperazine rings belonging to the epipolythiodioxopipreazines. In addition, the pentacyclic rearrangement involving the loss of the SMe· radical seems to occur in the presence of the unfused ring. Finally the product ions at m/z 635 and 591 seem to be the characteristic ions for chaetocochin A. Copyright © 2007 John Wiley & Sons, Ltd.