Host defence peptides are found in all classes of life and are a fundamental component of the innate immune response. Initially it was believed that their sole role in innate immunity was to kill invading microorganisms, thus providing direct defence against infection. Evidence now suggests that these peptides play diverse and complex roles in the immune response and that, in higher animals, their functions are not restricted to the innate immune response. In in vitro experiments certain host defence peptides have been demonstrated to be potent antimicrobial agents at modest concentrations, although their antimicrobial activity is often strongly reduced or ablated in the presence of physiological concentrations of ions such as Na+ and Mg2+. In contrast, in experiments done in standard tissue culture media, the composition of which more accurately represents physiological levels of ions, mammalian host defence peptides have been demonstrated to have a number of immunomodulatory functions including altering host gene expression, acting as chemokines and/or inducing chemokine production, inhibiting lipopolysaccharide induced pro-inflammatory cytokine production, promoting wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response. Animal models indicate that host defence peptides are crucial for both prevention and clearance of infection. As interest in the in vivo functions of host defence peptides is increasing, it is important to consider whether in mammals the direct antimicrobial and immunomodulatory properties observed in vitro are physiologically relevant, especially since many of these activities are concentration dependent. In this review we summarize the concentrations of host defence peptides and ions reported throughout the body and compare that information with the concentrations of peptides that are known have antimicrobial or immunomodulatory functions in vitro.