Two cell cycle-specific temperature sensitive (ts) mutants of mammalian cell lines, AF8 and K12, are known to arrest in G1 when shifted to the non-permissive temperature. We have determined the entry into S of both AF8 and K12 cells in five different growth conditions, namely: (1) quiescent sparse cultures stimulated to proliferative by serum; (2) quiescent dense cultures stimulated by serum; (3) quiescent sparse cultures stimulated by trypsinization and replating; (4) quiescent, dense cultures stimulated by trypsinization and replating; and (5) mitotic cells collected by mitotic detachment. In addition, for each cell line and for each different growth condition, we have determined the shift-up time, i.e., the time at which a shift-up to the nonpermissive temperature no longer prevents the entry of cells into S. In no case did K12 or AF8 enter S at the nonpermissive temperature. At the permissive temperature, the average time of entry into S varied in different growth conditions, and so did the shift-up time. However, in both cell lines, the distance of the average shift-up time from the average time of entry into S was remarkably constant, regardless of the growth conditions. i.e., 1.8 hours in K12 and 8.6 hours in AF8.