Five temperature-sensitive growth mutants of the hamster cell line BHK-21 were tested for the ability to support adenovirus 2 multiplication at 39 degrees and 33 degrees. Wild-type BHK-21 and mutants ts 422E and ts BCH yielded comparable amounts of virus at 33 degrees and 39 degrees, whereas in three other mutants, ts T22, ts T23, and ts AF8, virus production at 39 degrees was reduced to about 1% of that at 33 degrees. Virus yield in the three mutants was not reduced because of a delay in virus production; for all cells tested maximal virus yield at 39 degrees was obtained by 40-50 hr after infection. Normal yields of infectious virus were not obtained from ts AF8 even with a very high multiplicity of infection. In contrast, the virus yield from ts T22 and ts T23 was multiplicity-dependent. Shiftup experiments demonstrated that in ts AF8, a cell cycle mutant which at 39 degrees becomes arrested in G1, virus multiplication was thermosensitive for the first 40 hr of infection. In ts T22 and ts T23, the thermosensitivity was only for the first 3-4 hr of the infection. In all three mutants viral DNA synthesis was reduced by at least 95% at the higher temperature. The cell function specified by the ts AF8 mutation seems to be required for the early period of adenovirus 2 replication, after virus entry into the cell but before the onset of viral DNA replication.