Interferon (IFN)-ß is reported to have more potent antitumor effects than IFN-α. The aim of this study was to compare the synergistic antitumor activity of both IFNs when combined with gemcitabine on cultured pancreatic cancer cells expressing various levels of IFN receptor. The growth-inhibitory effects of IFN-α and IFN-ß in combination with gemcitabine on three human pancreatic cancer cell lines (BxPC-3, MIAPaCa-2, Panc-1) were evaluated by MTT assay and isobolographic analysis. We also correlated their growth-inhibitory effects with the expression status of type I IFN receptor type 2 (IFNAR2). Western blot analysis indicated strong expression of IFNAR2 in BxPC-3 and MIAPaCa-2, but weak expression in Panc-1. The growth-inhibitory effect of gemcitabine was enhanced synergistically by IFN-α in BxPC-3 and MIAPaCa-2, but not in Panc-1. IFN-ß exhibited more potent synergistic growth-inhibitory effects with gemcitabine in BxPC-3 and MIAPaCa-2 compared to IFN-α, and also synergistic enhancement in Panc-1. In conclusion, our results indicated that the growth-inhibitory effect of IFN-ß with gemcitabine was synergistic not only in pancreatic cancer cells with strong expression of IFNAR2, but also in those with weak expression of IFNAR2.