We have further characterized a temperature-sensitive mutant of Chinese hamster lung fibroblasts in tissue culture with a defect in RNA metabolism. The mutant phenotype is reflected in transcription in crude extracts or in isolated nuclei, when these are made from cells shifted to the nonpermissive temperature; however, differential heat inactivation between mutant and wild-type extracts cannot be demonstrated with cell-free systems. We tentatively conclude that the mutation may affect initiation of transcription which cannot be observed in our in vitro systems. Partially purified RNA polymerase I, II, and III fractions are indistinguishable from wild type. A temperature shift does not affect transcription by RNA polymerase III measured with intact cells or by nuclear run-on experiments. The nuclear run-on and other experiments suggest that RNA polymerase II-dependent transcription is inhibited before RNA polymerase I-dependent transcription. This conclusion is also supported by Northern analyses of selected mRNAs in nonsynchronized and synchronized cells after a shift to the nonpermissive temperature.