Type I interferons have an important role in modulating innate and acquired immune responses to viral infections and cancer, and also exhibit some direct antiproliferative activity. As an example, interferon alpha2 (IFNa2) is indicated for the adjuvant treatment of several systemic and lymphatic cancers, including leukaemia, lymphoma, Kaposi’s sarcoma and melanoma. Realising that the metastatic dissemination of cancers via the blood and lymph accounts for 90% of cancer-related deaths, our group has an interest in maximising therapeutic drug targeting towards lymphatic sites of cancer metastasis. To this end, we previously showed that PEGylated IFNα2 displays significantly higher lymphatic exposure than native recombinant IFNα2 and significantly inhibits the growth of lymphatic metastases in a mouse model of lymph-metastatic breast cancer more avidly than native IFNα2. Here, we therefore sought to identify PEGylation strategies that optimally enhance the systemic and lymphatic exposure, and activity of IFNα2 in a thoracic lymph duct cannulated rat model. The results showed that conjugation of IFNα2 with a single linear 5 or 20 kDa PEG chain provided a superior balance between plasma and lymphatic exposure and activity over existing PEGylated and unmodified versions of IFNα2 used clinically after a single subcutaneous dose. Specifically, the PEG5 construct displayed similar plasma exposure to ‘IFN activity’ (IU per ml plasma) as the 60 kDa PEG40 clinical product PEGASYS, while the PEG20 construct exhibited 3 fold higher plasma exposure to IFN activity when compared to both the 12 and 40 kDa PEGylated clinical products PEG-Intron and PEGASYS. The PEG5 and PEG20 constructs showed between 50 and 400% higher lymphatic exposure to IFN activity when compared to both PEGylated clinical products. These results suggest that adjuvant immunotherapy of systemic and lymphatic cancers with IFNα2 can be enhanced through the use of these more optimal alternative PEGylated variants.