Cytoplasmic extracts of untreated cultures of a virus-resistant mutant of mouse 3T6 cells, designated 3T6-VrB2, contain two double-stranded, RNA-activated enzyme activities associated with interferon action. These are the synthesis of a low molecular weight oligonucleotide inhibitor of cell-free protein synthesis from ATP, and the phosphorylation of a 67,000 dalton polypeptide by transfer of the gamma phosphate of ATP. Basal levels of both enzyme activities are detectable in extracts of untreated parental 3T6 cells, and are greatly enhanced upon interferon pretreatment. A procedure was developed, using a nonlonic detergent to effect cell lysis, which allowed the analysis of the protein kinase activity from as few as 2 × 107 cells. Using this procedure, direct proportionalities were demonstrated between the concentration of interferon to which 3T6 cells were exposed, and both the level of protein kinase activity and the magnitude of the antiviral state were established in these cells. Furthermore, untreated cultures of 3T6-VrB2 exhibited both an antiviral state and an intracellular protein kinase activity equal to that of cultures of the parental 3T6 cells pretreated with a single concentration of mouse interferon.