The mechanism of the failure of enterovirus type 70 to replicate at a nonpermissive temperature (39 degrees C) was investigated, and the following results were obtained. (i) Viral RNA synthesis was not observed at 39 degrees C in LLC-MK2 cells, in accordance with our previous findings with primary monkey kidney cells (Miyamura et al., Intervirology 9:206-213, 1978). (ii) Shutoff of host cell macromolecular synthesis by virus infection was as efficient at 39 degrees C as at a permissive temperature (33 degrees C). This inhibitory effect similarly occurred even in the presence of guanidine hydrochloride. (iii) Viral protein synthesis proceeded in vivo at the nonpermissive temperature, and the rate of the protein synthesis was higher than that at the permissive temperature under the conditions in which sufficient viral mRNA had been accumulated. This was also confirmed by analyzing the intracellular proteins synthesized at the nonpermissive temperature by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which identified them as virus-specific proteins. (iv) When infected cells were incubated at 39 degrees C and then transferred to 33 degrees C, viral RNA synthesis took place even in the presence of cycloheximide. (v) Furthermore, in experiments performed with an in vitro cell-free assay system, viral polymerase activity was found in the membrane-bound preparation extracted from infected cells which had been incubated at 39 degrees C in the presence or absence of guanidine hydrochloride. These results indicate that early translation of mRNA proceeds normally at the nonpermissive temperature and that the temperature-sensitive defect resides in the transcriptional stage.