The effect of the simultaneous use of 0.025% levocabastine hydrochloride eye drops (levocabastine) and 0.1% pemirolast potassium ophthalmic solution (pemirolast) on experimental allergic conjunctivitis in rats was investigated. Levocabastine and pemirolast significantly inhibited allergic conjunctivitis compared with the control group when separately administered. In addition, the simultaneous use of both drugs inhibited allergic conjunctivitis more potently than the original activity of levocabastine or pemirolast. Furthermore, the simultaneous use of levocabastine and pemirolast also significantly inhibited increased vascular permeability induced by antigen compared with levocabastine or pemirolast alone, respectively. Levocabastine and pemirolast inhibited histamine release from the rat conjunctiva in correlation with a decrease in histamine content in tears. When levocabastine and pemirolast were simultaneously applied to the eyes, histamine release from the conjunctiva was greater than for the original activities of both drugs. Similar to histamine release from the conjunctiva, the histamine content in tears induced by the simultaneous use of both drugs was significantly decreased compared with levocabastine and pemirolast alone, respectively. A potentiating effect induced by the simultaneous use of levocabastine and pemirolast may be attributable to the antihistaminic activity of levocabastine and histamine release inhibition by levocabastine and pemirolast.