alpha-Interferon (IFN-alpha) was produced by either peripheral blood lymphocytes or by monocytes and purified by an anti-IFN-alpha affinity column. When these preparations were analyzed by reversed-phase HPLC, a difference in the distribution of IFN-alpha subtypes from the two cell types was found. While the two major subtypes of IFN from induced lymphocytes had apparent molecular weights of 20K and 21K, monocytes produce an additional subtype of molecular weight 26K in large quantities (50%). This subtype had greater activity on human cells than on bovine cells in comparison to other IFN-alpha subtypes.