β-Defensins are natural peptide antibiotics whose immunomodulatory functions are poorly understood. In the present study, macrophages were found to migrate to human β-defensins (HBD)-1 to -4 using Gαi proteins as well as MAPK ERK, p38 and JNK as signal transducers. In addition, mast cells responded to HBD-1 to -4 with calcium fluxes as well as chemotaxis, which increased upon stimulation with IgE plus antigen or ionomycin. In contrast, human β-defensins were unable to induce migration of memory lymphocytes and dendritic cells (DC). Similar to HBD, the murine β-defensin (mBD)-8 mobilized macrophages and lacked the ability to recruit memory T cells. These findings were unexpected as CCR6 on memory T cells and DC has been previously observed to be a receptor for human β-defensins. In support of our findings, however, RBL-2H3 as well as 300.19 cells stably expressing CCR6 proved to be unresponsive to HBD-2 and -3. Intriguingly, our observation of a PKC-independent homologous desensitization between HBD-1 to -4 suggests a common receptor for HBD. In summary, chemoattraction of macrophages and mast cells is evolutionary conserved within the β-defensin family despite a considerable sequence variation and distinct antimicrobial activities. However, CCR6 is not a functional receptor for β-defensins.