Even though skin and oral mucosae are continuously in contact with commensal and opportunistic microorganisms, they generally remain healthy and uninflamed. Host defense peptides (HDPs) make up the body's first line of defense against many invading pathogens and are involved in the orchestration of innate immunity and the inflammatory response. In this study, we investigated the effect of two salivary HDPs, LL-37 and Hst1, on the inflammatory and antimicrobial response by skin and oral mucosa (gingiva) keratinocytes and fibroblasts. The potent antimicrobial chemokine CCL20 was investigated and compared with chemokines CCL2, CXCL1, CXCL8, and CCL27 and proinflammatory cytokines IL-1α and IL-6. Keratinocyte-fibroblast cocultures showed a synergistic increase in CCL20 secretion upon Hst1 and LL-37 exposure compared to monocultures. These cocultures also showed increased IL-6, CXCL1, CXCL8, and CCL2 secretion, which was IL-1α dependent. Secretion of the antimicrobial chemokine CCL20 was clearly IL-1α independent. These results indicate that salivary peptides can stimulate skin as well as gingiva cells to secrete antimicrobial chemokines as part of the hosts' defense to counteract infection.