The synthesis of infectious virus is reduced by 99% in adenovirus type 2-infected KB cells maintained at 42 C in suspension. Studies to delineate the steps in virus biosynthesis blocked at 42 C revealed the following. (i) The inhibition of ribosomal ribonucleic acid (RNA) synthesis and the block in the conversion of ribosomal RNA precursors to ribosomal RNA which normally occur in infected cells at 37 C was accentuated at 42 C. (ii) The same species of early and late viral messenger RNA were synthesized at 37 and 42 C. (iii) The shift from host cell deoxyribonucleic acid (DNA) synthesis to viral DNA replication occurred earlier at 42 C than at 37 C. These findings indicate that the thermosensitive block(s) in virion formation does not occur at the level of viral DNA replication or transcription of viral mRNA but probably involves the synthesis of late viral proteins or the maturation of the virion.