Four novel trypsin-like S1A peptidase transcripts (LsTryp2-5) from the marine parasitic copepod Lepeophtheirus salmonis were characterised based on analyses of 1918 expressed sequence tags from two adult female libraries. In addition, one previously described salmon louse trypsin, LsTryp1, has been further characterised. The five peptidases possessed all residues typically found in trypsins in correct sequence contexts. Interestingly, two cysteine residues, possibly involved in a disulphide bridge not previously reported in trypsins are conserved in all louse trypsin sequences. Phylogenetic analyses showed that the five louse peptidases form a monophyletic group with other crustacean trypsins (Brachyurin Ts). Quantitative PCR analyses demonstrated increased transcript levels from planktonic to early host-attached stages and from preadult to sexually mature adult stages. Furthermore, sex-specific differences in transcription regulation were found. In situ hybridisation demonstrated that all five trypsin-like peptidases are transcribed throughout the undifferentiated midgut, indicating a digestive function. The sequence characteristics, histological localisation and transcript regulation suggest that LsTryp1-4 encode typical digestive trypsins. LsTryp5, however, showed some sequence and regulatory peculiarities that rendered its function less clear. Our findings support earlier suggestions for the function of the midgut cells and suggest the existence of an additional undifferentiated cell-type.