Interferon (IFN) production induced by either Newcastle disease virus (NDV) or Sendai virus was compared in 10 different rat cell lines. Although there was variation in the IFN titers produced, NDV proved to be the best inducer in each cell line with optimum IFN yields occurring with a multiplicity of infection (MOI) of 1.0. A new continuous rat fibroblast cell line (CD) produced high titers of IFN similar to those reported for other high producers; while Ratec cells were shown to be the most sensitive to the antiviral activity of rat IFN. Partial purification and characterization of IFN produced in CD cells was accomplished by column chromatography. Four sorbents with varying modes of action for binding the IFN (Affi-Gel 202, Poly(U)-Sepharose 4B, CM-Sepharose CL-6B, and Phenyl-Sepharose CL-4B) were compared. The Phenyl-Sepharose CL-4B matrix proved to be the most successful for purification of rat IFN; one passage through this column, increased the specific activity more than 100-fold, with a concomitant recovery of 90%-95% biologic activity. The binding characteristics of rat IFN on each of the column matrices, however, demonstrated differences between the physicochemical nature of CD rat IFN and murine and hamster IFNs.