It has been reported that antihistamines do not fully modify symptoms of allergic conjunctivitis in clinical settings, suggesting that histamine is not the only contributor to symptom generation in the disease. However, in the majority of experimental allergic conjunctivitis models, antihistamines are very effective in the reduction of symptoms. In the present study, we used our recently developed guinea pig model of allergic conjunctivitis and evaluated whether involvement of histamine in the induction of symptoms of allergic conjunctivitis is altered by multiple antigen challenges. Guinea pigs were sensitized by intraperitoneal injection of Japanese cedar pollen extracts adsorbed on aluminum hydroxide gel, and then challenged by dropping a pollen suspension without the adjuvant on each eye once a week until the 15th challenge. The magnitude of the conjunctivitis intensity score (CIS), itch-associated scratching response and albumin leakage were found to increase with repeated challenges. At the 1st-3rd challenges, histamine H(1) receptor antagonist, mepyramine (10 mg/kg, p.o.), strongly reduced all these symptoms. However, symptoms at the 5th-15th challenges were not inhibited by mepyramine. On the other hand, a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (10 mg/kg, i.v.), potently inhibited the increase of CIS and albumin leakage at the 15th challenge. In conclusion, histamine involvement in the induction of conjunctivitis symptoms in our model was diminished by multiple antigen challenges. The allergic conjunctivitis at the chronic stage is partly mediated by nitric oxide (NO) derived from NOSs that may be activated by mediators other than histamine. The histamine-independent allergic conjunctivitis may be useful for analyzing mechanisms underlying chronic conjunctivitis.