Both mast cells and eosinophils were implicated in the pathophysiology of allergic conjunctivitis; however, the potential role of eosinophils in an early phase of allergic reaction has not been elucidated. The aim of this study was to assess the relation between clinical symptoms and sequence of mast cells and eosinophils specific mediators release into tear fluid during conjunctival allergen provocation. Patients with grass pollen rhinoconjunctivitis (n = 38) and healthy volunteers (n = 10) were challenged with increasing doses of allergen applied on the conjunctiva. The clinical symptoms were assessed by clinical score. Tear fluid was collected from 12 patients before provocation, at 20 and 40 minutes after positive response. Tryptase and eosinophil cationic protein (ECP) were measured using UniCap and 15-hydroxyeicosanoid acid (15-HETE) with a specific immunoassay. All allergic patients (but no control subjects) had a positive clinical response to the challenge. In 1 patient symptoms appeared after 50 BU/mL of grass allergen administration, in 3 patients symptoms appeared after 500 BU/mL (7.9% of patients), in 14 patients symptoms appeared after 1600 BU/mL (36.8%), and in 20 patients symptoms appeared after 5000 BU/mL (52.6%). The allergen dose was not correlated with the skin-prick test diameter. The mean tryptase concentration increased at 20 minutes from "nondetectable" to 5.89 ± 1.97 micrograms/L and then decreased to 1.77 ± 1.07 micrograms/L (n = 12; p < 0.05) at 40 minutes. ECP concentration was not changed at 20 minutes but increased at 40 minutes from 1.38 ± 0.98 micrograms/L before provocation to 10.61 ± 7.78 micrograms/L (n = 7; p < 0.05). There was no change in 15-HETE release. Both mast cells and eosinophils are activated during allergic reaction in conjunctiva and activation of eosinophils is preceded by activation of mast cells.