An initial bioinformatics investigation followed by cloning and sequencing analysis, has led to the identification of three novel members (omDB-2, omDB-3, omBD-4) of the beta-defensin family in rainbow trout (Oncorhynchus mykiss). The contiguous sequences could be translated to give predicted peptides of 62 (omDB-2), 63 (omDB-3) and 68 (omDB-4) amino acids (aa) in length, with mature peptides of 43 (omDB-2), 39 (omDB-3) and 42 (omDB-4) aa, with no obvious proregion present. Analysis of the gene organization found that all three new genes contained three exons divided by two introns, as seen in defensin genes of other fish species. Constitutive expression of all the trout defensins was detected by RT-PCR in a wide range of mucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout beta-defensin genes were induced in head kidney primary leucocyte cultures at 4h post-stimulation, with omDB-1 and omDB-3 particularly highly expressed. These data suggest that beta-defensins are likely an important component of the innate defences of fish, and reveal an added level of antimicrobial peptide complexity in fish to that known previously.