Conditions for the formation of hybrids between polyoma DNA immobilised on nitrocellulose filters and RNA extracted from lytically infected mouse cells were established. At 39°0 in 50% formamide: 50% 2xSSO, the reaction is sub optimal with respect to initial kinetics, but degradation of RNA is minimal and specificity is retained. RNA can be eluted from hy9rids using 50% DMSO: 50% water at 42°0. ii) The size of polyoma specific RNA (PyRNA) prepared in this way from lytically infected mouse cells was examined. At all stages in infection, nuclear PyRNA is heterogeneous in size, and contains molecules larger than a single strand of viral DNA. Such molecules contain host sequences and are likely to have been transcribed from an integrated viral genome. In the cytoplasm, early RNA made in the presence of cytosine arabinoside consists largely of a 0.1 x 106- species. Late in infection the major cytoplasmic species is 1 x 106 daltons in mass. Hybridisat1on-competition experiments indicate 40% of the sequences present late in the cytoplasm:'are also present early. When DNA synthesis is inhibited during the period in which it is rising to its maximum, then the resulting cytoplasmic RNA resembles early RNA both in size and by its behaviour: in hybridisation-competition experiments./ The proportion of late sequences in the nucleus is also reduced by 50%. Late RNA synthesis is therefore coupled to DNA synthesis. iii) Cordycepin prevents the appearance of PyRNA in the cytoplasm. PyRNA was demonstrated to contain poly A rich sequences. Several techniques were attempted as a means of purifying PyRNA for use in in vitro protein synthesising / systems. Polysome dissociation using puromycin or EDTA was found to be unsatisfactory as a means of separating PyRNA from ribosomal sub-units. The immunoprecipitation of polysomes bearing nascent polyoma coat protein was found to be unsatisfacto17 as RNA produced in this way was degraded. The biological activity of PyRNA eluted from hybrids has not yet been demonstrated.