Background: The modulation of T cell responses by natural peptides is of potential use in the treatment of allergic diseases and reduces the risk of IgE-mediated reactions. The objectives of this study were to evaluate the allergenicity of depigmented, pepsin-digested fragments of Oela europaea and Phleum pratense extracts and their capacity to induce T cell proliferation. Methods: Lyophilized O. europaea and P. pratense extracts were depigmented following standardized methods, digested with pepsin and fractionated by ultrafiltration; a fraction containing components in the molecular weight range of 1–10 kD was selected. The blood of 20 allergic individuals was used to conduct T cell proliferation studies. Results: Pepsin-digested fragments of both pollen extracts lost more than 95% of their IgE-binding capacity by ELISA inhibition. Greater proliferation indexes than their respective native extracts were obtained in 55% of O. europaea and in 95% of P. pratense allergic patients. Conclusions: This study demonstrates that pepsin-digested allergenic fragments of depigmented extracts can be produced in a standardized, reproducible manner. These fragments are significantly less allergenic and retain, or even increase, their capacity to induce T cell proliferations.