Purpose - To investigate the expression of human β-defensins (hBDs) by human corneal epithelium and determine the effects of proinflammatory cytokines on expression of human β-defensin (hBD)-2 by human corneal epithelial cells (HCECs) in culture. Methods - RNA was extracted from corneal epithelial cells scraped from cadaveric corneas and from cultured HCECs, and RT-PCR was performed to detect hBD-1, -2, and -3 mRNA. To study the effects of proinflammatory cytokines on expression of defensin, HCECs were cultured and then exposed to interleukin (IL)-1β or tumor necrosis factor (TNF)-α for up to 36 hours, with a range of concentrations (0.01–100 ng/mL). In some experiments, cells were pretreated with various cell signaling pathway inhibitors before the addition of IL-1β. At the end of the incubations, the cells were harvested for RT-PCR and the culture media collected for the detection by immunoblot analysis of secreted defensin peptide. Results - All epithelial tissue collected from cadaveric corneas expressed mRNA for hBD-1. hBD-2 was detectable in two of eight donors corneas, whereas hBD-3 was detected in five. All primary cultures of HCECs expressed hBD-1 and -3. A faint band for hBD-2 was detectable in three of eight cultures. Cultures of simian virus (SV)40-transformed HCECs always expressed hBD-1 and -3, but did not express hBD-2 under control conditions. IL-1β and TNFα each stimulated the expression of hBD-2 in HCECs and were more effective in combination than alone. The effects of IL-1β were concentration- (maximal at 10 ng/mL) and time-dependent (maximal at 12 hours and 24 hours for hBD-2 mRNA expression and protein secretion, respectively). The upregulation of hBD-2 mRNA persisted for at least 24 hours after removal of IL-1β. The NFκB inhibitors pyrrolidinedithiocarbamate (PDTC; 100 μM), caffeic acid phenethyl ester (CAPE; 90 μM), and MG-132 (25 μM), blocked IL-1β–stimulated expression of hBD-2. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (5 μM) and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (25 μM) partially blocked (by 47% and 59%, respectively) the effect of IL-1β. However, PD98059, an ERK inhibitor, had no effect. Genistein (50 μM) and dexamethasone (1 μM) also partially blocked (by 26% and 28%, respectively) the effect of IL-1β. Conclusions - Human corneal epithelium expresses hBD-1 and -3. hBD-2 is not typically present, but its expression can be stimulated by proinflammatory cytokines such as IL-1β, acting through mitogen-activated protein (MAP) kinase and nuclear factor (NF)-κB pathways. Because IL-1 is known to be increased at the ocular surface after injury, the current observations provide a mechanism to explain the previous finding that hBD-2 is upregulated in regenerating corneal epithelium. Cytokine stimulation of hBD-2 expression most likely provides additional protection against infection and raises the possibility that this defensin in particular may be involved in the wound-healing response, per se.