Purpose. To characterize phenotypically the primary epithelial cells obtained from normal bulbar conjunctival biopsy specimens and a human conjunctival epithelial cell line (Wong-Kilbourne derivative of Chang conjunctiva; ChWK) with regard to their immunostimulatory function. methods. The effects on expression of HLA-DR and costimulatory molecules (CD80, CD86, ICAM-1, and CD40) on normal conjunctival epithelial cells and ChWK treated with various cytokines (IFN-γ, TNF-α, IL-4, and IL-13) were determined using flow cytometry and confocal microscopy. results. Epithelial cells were successfully grown from conjunctival explants and cultures passaged three times, while retaining their cell surface markers. At least 97% of primary epithelial cells (n = 10) and more than 96% of ChWK cells (n = 10) were cytokeratin positive by flow cytometry and immunocytochemistry and demonstrated epithelial cell morphology. Both primary conjunctival epithelial cells and ChWK had a low basal expression of HLA-DR and ICAM-1, and both were upregulated by IFN-γ. For ChWK cells, CD80 and CD86 were constitutively expressed at low levels. CD80 was significantly upregulated after IFN-γ treatment (P = 0.043), whereas IL-4 induced a significant upregulation of CD86 (P = 0.039). Treatment with IL-13 and TNF-α did not induce significant effects. conclusions. The ability of conjunctival epithelial cells to express costimulatory molecules suggests a proinflammatory role for conjunctival epithelial cells.