Summary Using conventional procedures of extraction with diethyl ether to remove phenol, the infective poliovirus RNA titer was found to be 5 to 100 times greater when the virus stock was diluted about one-hundredfold into certain diluents prior to treatment with phenol (560 mM) than when undiluted stock was treated. Diluents giving this result were calcium-free, magnesium-free, mildly alkaline (pH 7–11) solutions, notably, e. g., 10 mM NaHCO3. The milieu afforded by such diluents was not necessary for the treatment with phenol but was necessary primarily to prevent “inactivation” of the poliovirus RNA by the ether; however, even without exposure to ether and with no extractions to remove phenol, mildly acid conditions or a moderate concentration of Ca++ resulted in some loss of infective RNA titer. Assay of ethereal phase detected no infectivity. Even with added base and chelating agent, extractions of phenol-treated essentially undiluted virus stocks with ether resulted in large loss of infective RNA titer. Extractions with benzene in place of ether had little or no effect on RNA titer using diluted systems; but with undiluted systems, a large titer loss was found. Treatment with ether without extractions with ether also inactivated RNA. Using heat shock (70° C, 20 seconds) in place of phenol to obtain infective RNA, the nature of the diluent required to give high infective RNA titer was very different from that required for the prephenol dilution when the conventional extractions with ether were employed.