Abstract Thermosensitive mutants of echovirus 12 were isolated after mutagenization with 5-fluorouracil. Twelve of them were characterized in terms of the physiological defects that appear under restrictive conditions. At the nonpermissive temperature only 2 of the 12 mutants are able to synthesize viral RNA and protein in near-to-wild type amounts; however, no viral or subviral particles could be detected in cells infected with these two mutants. The remaining 10 mutants were found to be impaired in viral RNA synthesis. Six of the twelve mutants produced virus particles which were more thermolabile in vitro than those of the wild type; it is concluded that they carry a mutation in the gene(s) coding for structural protein. All of them belong to the class of mutants which were defect in viral RNA synthesis. These results suggest an involvement of structural protein in viral RNA synthesis.