Interferon was produced by interaction of UV inactivated Influenza A virus, Melbourne strain, and primary kidney tissue cultures of rhesus monkey, African green monkey, calf and rabbit kidney, human embryo, bovine embryo, hamster embryo, chorioallantoic membrane of chick embryo and continuous passage lines of dog and pig kidney. The activity of various interferons was determined in homologous and heterologous cell systems challenged with 106.5 EID50 of Melbourne virus. The results confirm and extend published observations concerning species specificity of chick embryo and mammalian cell-derived interferon. Interferon made in human embryonic kidney cells exhibited the broadest heterologous activity. Rabbit kidney cells were most sensitive for detecting heterologous interferon from mammalian sources. Pig kidney and hamster embryo cultures, insusceptible to influenza virus, produced interferon. Sensitivity of assay method was increased by using challenges of 30 or less TCID50 of Melbourne virus. Monkey kidney and rabbit kidney interferons were then detected in both homologous and heterologous kidney cells. Thus, species specificity of mammalian interferon did not appear to be absolute, and interferon-induced cellular resistance may be overcome by an overwhelming dose of challenge virus.