TWO species of form A (or I) RNA polymerase have been identified in eucaryotic cells and there is evidence that this a-amanitin-insensitive activity exists as two discrete pools: a pool of 'free' activity, which is identified by its ability to transcribe poly [d(A-T)] in the presence of actinomycin in vitro, and a pool of enzyme in the form of a transcription complex ('engaged') which is unaffected by inhibitors of initiation of RNA synthesis.I. The principles underlying and the practical application of the technique used to define the pool of 'free' RNA polymerase activity have been analysed in considerable detail. On the basis of actinomycin titrations of poly[d(A-T)]dependent activity in isolated organelles, it is concluded that a pool of 'free' RNA polymerase A activity exists in mammalian nuclei which, under certain circumstances, is lost from nuclei during their isolation. The evidence presented suggests that nucleoli, resolved from nuclei by the classical sonication technique, contain form A polymerase exclusively in the transcription complex form.2. Different techniques used to solubilise RNA polymerase activity from nucleoli are shown to give rise to different proportions of the two formA RNA polymerase species (A1 and AII, as defined by their differential elution from phosphocellulose) : whereas low-ionic-strength extraction gives rise to form AII, high-salt, sonication extracts contain predominantly the form A1 enzyme. It is shown that the sonication technique results in the conversion of form A11 to form AI. By a careful appraisal of the products of these procedures and a novel polymerase solubilisation technique, it is concluded that RNA polymerase A11 is the 'engaged' form of the enzyme found in the transcription complex.3 . Making use of the finding that the 'free' form of the enzyme is lost to the cytoplasmic fraction on nuclear isolation, this activity has been characterised without the requirement for solubilisation techniques which might result in the conversion of one form to another: the 'free' species is shown to be form A1 RNA polymerase.4. These conclusions that two discrete pools of formA RNA polymerase activity contain different species of the enzyme are briefly discussed in the light of other published information concerning their subunit structures and their potential role in the expression of the ribosomal RNA coding sequences.The form A (or type I) DNA-dependent RNA polymerase activity is considered to be localised primarily within the nucleolus [l] and is thought to be responsible for the expression of the ribosomal RNA coding sequences in eucaryotic cells [2,3]. It has been known for some time that at least two species of form A polymerase may exist : this apparant heterogeneity was first described for an activity derived from rat liver which was resolved into two components by phosphoEnzyme. RNA polymerase or nucleoside triphosphate: RNA nucleotidyltransferase (EC 2.7.7.6).cellulose chromatography [4]. These activities are referred to as forms A1 and A11 according to their order...