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This paper describes a large-scale method for the solubilization and the separation of calf thymus A and B DNA-dependent RNA polymerase activities. This method is reproducible, gives high yields and can handle up to several kg of tissue. The solubilization method involved homogenization of the whole tissue, sonication a t high ionic strength, ammonium sulphate precipitation and protamine sulphate precipitation to remove nucleic acids. The separation of the two types of activities A (insensitive to a-amanitin) and B (inhibited by this compound) was achieved by differential adsorption to DEAE-cellulose using a batch procedure. Studies of the inhibitory effect of a-amanitin on the various fraction during the solubilization and the separation stages suggest very strongly that the two types of activities pre-existed in vivo and did not result from the solubilization procedure.The separation of several peaks of DNA-dependent RNA polymerase activities on DEAE-cellulose or DEAE-Sephadex columns [1,2] and the differential effect of the inhibitor a-amanitin on these peaks [2] suggested that animal cells contained multiple RNA polymerases with specific intranuclear localizations [3]. Definite proof of the existence of several distinct enzymes in animal cells requires the purification of these activities and the demonstration that they correspond to different proteins. To carry out such studies, for which milligram quantities of highly purified enzyme are required, it was necessary to work out a large-scale purification procedure since the content of DNAdependent RNA polymerase in animal tissues, on a weight basis, is at the most 'Ilo of that of procaryotic cells (see Conclusion). This paper describes the first steps of such a procedure, which can be applied to kilogram quantities of tissue and which results in the separation of two types of activity; type A which is insensitive to a-amanitin and type B which is completely inhibited a t very low concentrations of this compound. Preliminary reports of this work have been published [2,4].

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