Incubation of HeLa cells at 42 degrees C results in pronounced inhibition of the accumulation of 18S and 28S ribosomal RNA (rRNA) and non-heat shock polyadenylated messenger RNA (mRNA) in the cytoplasm. Accumulation of transfer RNA and 5S ribosomal RNA is not affected. Transcription of rRNA precursor is reduced to approximately 50% of the 37 degrees C rate after 10 min of hyperthermia and declines to 30% of the control rate after 1 hr. In contrast, the accumulation of mature rRNA in the cytoplasm is inhibited more than 95%. Quantitative hybridization experiments and Northern blot analysis detect little accumulation of rRNA precursor sequences in nuclei, suggesting that the majority of the rRNA that is synthesized is degraded. Heat stress at 42 degrees C was found to have little effect on transcription of most non-heat shock mRNAs. However, accumulation of individual non-heat shock mRNAs in the cytoplasm proceeds at reduced rates. These results indicate that the primary effect of elevated temperature on RNA metabolism in mammalian cells is inhibition of processing and/or transport. Despite this, steady-state levels of abundant and rapidly turning over mRNA species remain unchanged during prolonged heat stress. We find that the half-life of c-myc mRNA increases greater than twofold at 42 degrees C. Thus, 42 degrees C heat stress appears to inhibit both accumulation and turnover of non-heat shock mRNA.