Background: Several cytokines are involved in the recruitment and activation of inflammatory cells in ocular allergic diseases. The purpose of the study was to assay multiple cytokines and chemokines in tears, to compare subgroups of allergic conjunctivitis (AC) with controls, and in culture supernatants to determine whether conjunctival fibroblasts produce some of these cytokines. Methods: Fifty to one hundred microlitre tears were obtained from patients with active seasonal allergic conjunctivitis (SAC; n=12), vernal keratoconjunctivitis (VKC; n=18), atopic keratoconjunctivitis (AKC; n=6) and non-atopic controls (n=14). Primary conjunctival fibroblasts grown in vitro were stimulated with IL-4, IL-13 or TNF- alpha for 24 h. Cell-free tear and culture supernatants were assayed for IL-1 beta , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IFN- gamma , TNF- alpha , eotaxin, MCP-1 and RANTES using multiplex bead analysis. Induction of chemokine gene expression was determined by PCR. Results: IL-1 beta , IL-2, IL-5, IL-6, IL-12, IL-13, MCP-1 were increased in all tears groups compared with controls, with highly significant correlations between many of these molecules. In addition IL-4, IFN- gamma , and IL-10 were elevated in SAC and VKC, while eotaxin and TNF- alpha were only increased in VKC. IL-6, IL-8, MCP-1, RANTES and eotaxin were detected from fibroblasts cultures, and were all up-regulated by TNF- alpha . By PCR, fibroblasts expressed MCP-1 transcripts constitutively, whereas IP-10 and Mig were up-regulated by TNF- alpha . Conclusions: Differential cytokine levels support tears as a useful indicator of immune mechanisms occurring during AC. The striking similarities in chemokine profiles between tears and fibroblasts suggest these cells as likely sources of chemokines in tears.