We compared the expression of human α‐ and β‐defensins by various human tissues. mRNA for α‐defensins HNP1‐3, abundant in bone marrow, was detected in peripheral blood leukocytes, spleen and thymus by RT‐PCR, which revealed α‐defensins HD5 and HD6 only in the small intestine. In contrast, the pancreas and kidney expressed high levels of hBD‐1 and lower levels of this β‐defensin were found in many organs by RT‐PCR (salivary gland > trachea > prostate and placenta > thymus, testis, small intestine). hBD‐1 mRNA was produced constitutively by cultured normal human epithelial cells derived from the trachea, bronchi, small airways and the mammary gland. These largely non‐overlapping tissue distributions of human α‐ and β‐defensins suggest that hBD‐1 may be positioned to defend epithelial cells and mucosae from infection, whereas expression of HNP1‐3 in neutrophils and HD5 and HD6 in Paneth cells allows these α‐defensins to participate in systemic and small intestinal host defenses, respectively.