Human beta-defensins are antimicrobial peptides that may be critical in the innate immune response to infection. hBD1 and hBD2 are expressed in oral epithelial cells and are detected near the surface of oral tissue, consistent with a role in the epithelial protective barrier function. In this report, we examine secretion of beta-defensins in vitro and in biological fluid using ProteinChip(R) Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry. We show that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major secreted forms. These forms are both expressed and secreted under conditions anticipated from previous analysis of beta-defensin mRNAs; specifically, hBD1 is detected in culture supernatant from both unstimulated and stimulated cells, and hBD2 is detected only in stimulated cells. Identity of hBD1 and hBD2 was confirmed by immunocapture on the ProteinChip surface. Both peptides are also present in gingival crevicular fluid that accumulates between the tissue and tooth surface, although hBD1 is also found in several smaller forms suggesting extracellular proteolysis. This methodology offers several technical advantages for detection of defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size.