Allergen-specific CD4+ Th2 cells play an important role in the immunological processes of allergic asthma. Previously we have shown that, by using the immunodominant epitope OVA323-339, peptide immunotherapy in a murine model of OVA induced allergic asthma, stimulated OVA-specific Th2 cells, and deteriorated airway hyperresponsiveness and eosinophilia. In the present study, we defined four modulatory peptide analogues of OVA323-339 with comparable MHC class II binding affinity. These peptide analogues were used for immunotherapy by s.c. injection in OVA-sensitized mice before OVA challenge. Compared with vehicle-treated mice, treatment with the Th2-skewing wild-type peptide and a Th2-skewing partial agonistic peptide (335N-A) dramatically increased airway eosinophilia upon OVA challenge. In contrast, treatment with a Th1-skewing peptide analogue (336E-A) resulted in a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production. Our data show for the first time that a Th1-skewing peptide analogue of a dominant allergen epitope can modulate allergen-specific Th2 effector cells in an allergic response in vivo. Furthermore, these data suggest that the use of Th1-skewing peptides instead of wild-type peptide may improve peptide immunotherapy and may contribute to the development of a successful and safe immunotherapy for allergic patients.