Innate immune defense against microbial pathogens occurs by physical barriers, by recruitment of cells such as neutrophils, NK cells, and macrophages, and by secretion of molecules with antimicrobial activity. Such molecules are produced by various epithelia including skin. The importance of antimicrobial peptides has been shown in cathelicidin-deficient mice, which have increased susceptibility to skin infection by Streptococcus. Although keratinocytes increase cathelicidin expression upon injury, their role relative to neutrophil cathelicidin and their sites of peptide storage and activation have not been elucidated. Herein, it is reported that cathelicidin predominantly resides in granules of the superficial epidermis and partially localizes in lamellar bodies as determined by immunogold electron microscopy and immunoblot of lamellar bodies isolated from mice. In cultured keratinocytes, cathelicidin displays a granular distribution and partially localizes within the Golgi apparatus. Cathelicidin processing can be observed by western blot analysis in keratinocyte extracts but not in conditioned media. Further, fluorescent bacteria colocalize with cathelicidin in granules both intracellularly and at the cell surface. These observations illustrate the immune defense potential of keratinocytes acting directly through storage and processing of antimicrobial peptides.