The microheterogeneity of the paracelsin mixture broth of Trichoderma reesei was analysed using mass spectrometric methods, in particular high-energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). Based on the liquid secondary ion mass spectrum of the mixture, there are three main components, with molecular masses M and (M ± 14), together with two minor components of molecular weight (M ± 28). The high-energy CID tandem mass spectra of both the protonated and sodiated molecules yielded abundant and characteristic fragment ions, but of very different types. It was found that a paracelsin peptaibol in a mixture could be successfully sequenced based on the tandem mass spectra of its protonated and sodiated molecules or, alternatively, on the tandem mass spectra of its y 7 and b 13 fragment ions. A terminology for indicating these sequential peptide fragments is proposed. To determine the sequence of new analogues, tandem mass spectra of the y 7 , (y 7 ± 14), b 13 , (b 13 ± 14) and (MH ± 14) positive ions were also taken. Based on these experiments, four new paracelsin components (PA-F, PA-G, PA-H and PA-I) were sequenced successfully. The microheterogeneity of the mixture was found to be more pronounced than had been assumed previously. In these new analogues, besides positions 6 and 9, position 17 is also involved in the exchange. MS/MS studies on minor fragment ions, such as (b 13 -28) and (b 8 -14) show further microheterogeneity at positions 3, 5, 10 and 12, which increase the number of possible minor components.