A simple method for testing a variety of substances for antiviral activity against the pox group of viruses has been evolved in which serial dilutions of the substance in question were mixed with the virus and incubated in vitro before inoculation into the yolk sac of embryonated eggs. The viruses of canary pox, fowl pox and vaccinia were completely inhibited by 3,750, 7,500 and 15,000 units of commercial penicillin respectively when the virus was mixed with the penicillin and incubated in vitro before inoculation into chick embryos. An impurity or impurities present in commercial penicillin rather than penicillin itself was found to be responsible for the inhibition of canary pox virus in vitro. The antiviral activity of commercial penicillin against canary pox virus in vitro was inhibited by the presence of normal egg yolk, but was not significantly diminished when aqueous solutions were heated to 100 C for 10 minutes. Inoculation of 50,000 units of commercial penicillin prior to the inoculation of canary pox virus into the yolk sac of chick embryos significantly but not strikingly retarded the rate of death of the embryos.