In recent times there has been an enormous rise in resistance to synthetic antibiotics as well as an increase in the virulence of bacteria, the so-called "superbugs". This problem has catalyzed a search for novel molecules to fight bacteria, which in turn relies on a better understanding of the molecular basis of the immune response. β-defensins are a class of small, cationic, cysteine-rich antimicrobial peptides expressed by humans and other animals to act against incoming pathogens. As well as their antimicrobial, properties, β-defensms also act as chemokmes, recruiting cells to the sites of infection. Here the relationship between the tertiary structures of β-defensin analogs and their chemotactic activities has been investigated using ion mobility-mass spectrometry (IM-MS) and biochemical assays. A panel of derivatives of the murine β-defensin Def b14 has been formed and the ability of these peptides to chemoattract the receptor CCR6 has been assessed in vitro. The derivatives can be divided into two groups, those with chemotactic activity equal, to that of the unmodified parent peptide, and those whose chemotactic activity has been lost upon modification. Analysis by ion mobility-mass spectrometry reveals the conformational preferences of these peptides upon ionization from, different solvents. Under denaturing conditions, the chemotactic peptides adopt more compact conformations in the gas-phase at higher charge states than those which are inactive. While the conditions of these experiments are not akin to the environment around the receptor in vivo, this technique provides an in vacuo method for distinguishing between the different chemotactic activities of β-defensin derivatives. © 2010 American Chemical Society.