Temperature-sensitive Chinese hamster cells (K12) have been shown to be defective for the initiation of new rounds of DNA replication when incubated at the restrictive temperature (40.5 degrees). By temperature shift experiments with synchronous cultures, we have marked out the step at which the mutation is expressed as the four hours preceding the initiation of DNA synthesis. The block imposed by the mutation has been shown to be irreversible. In order to approach the biochemical characterization of the temperature-sensitive function in K12 cells, we have analyzed the cellular proteins synthesized under permissive (35 degrees) and restrictive temperatures. The synthesis of three polypeptides is markedly enhanced when K12 cells are incubated at 40.5 degrees. One of them (band B) has turned out to be a useful biochemical marker of the expression of K12 mutation since its synthesis is not affected in other ts-mutants or in hybrids in which K12 mutation is complemented. In addition, the alteration in band B synthesis is irreversible and occurs during the same stage of the cell cycle at which the mutated function is expressed.