Antimicrobial peptides (AMPs) are effector molecules of innate immunity. Dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is produced constitutively by the eccrine sweat glands and secreted into sweat. In this study, we investigated the proteolytic processing, site-specific expression, and stability of DCD peptides in eccrine sweat. Using surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and reversed-phase high-pressure liquid chromatography analysis, we identified in eccrine sweat 14 proteolytically processed DCD peptides. Semiquantitative SELDI-TOF-MS analysis indicated that processing of DCD-1L is individually different, but generates a few dominant peptides. At body sites with a high probability for contact with pathogenic microorganisms, a high amount of antimicrobial active DCD peptides was detected in sweat. Furthermore, we show that the secretion rate of DCD is constant during a period of prolonged sweating and that DCD peptides are stable in sweat over several hours. Other known AMPs like the human cathelicidin LL-37 and alpha- or beta-defensins were not detected in significant quantity in eccrine sweat. Owing to the durable and abundant presence, DCD-derived peptides contribute to the first line of defense by building a constant barrier that overlies the epithelial skin.