We have previously shown that >90% of 86.1 cells, a murine cytolytic T lymphocyte (CTL) cloned line which is solely dependent on T cell growth factor (TCGF) for continuous growth in vitro, accumulates in the GI phase of the cell cycle after transfer into culture medium containing no TCGF. Moreover, when such quiescent cells are exposed again to TCGF, >85% reenter the S phase and subsequently divide in a relatively synchronous fashion. In this study, the regulation of the rate of cell cycle progression of quiescent B6.1 cells after exposure to TCGF was analyzed using two complementary DNA staining techniques, namely, t h e propodium iodide method (to enumerate cells entering the S phase) and t h e Hoechst 33342-bromodeoxyuridine substitution technique (to enumerate cells which have gone through mitosis). After TCGF addition, quiescent 66.1 cells resumed DNA synthesis and divided after a lag phase of 10 and 20 h, respectively. The duration of the lag phase was found to be dependent on the length of time during which quiescent 86.1 cells had been deprived of TCGF, but was independent of the concentration of TCGF used for restimulation. In contrast, the proportion of cells responding to TCGF as well as the rate of their first passage through mitosis was dependent on TCGF concentration. The presence of TCGF for at least 6 h was required for a maximal response. Moreover, direct evidence was obtained that TCGF by itself was able to stimulate proliferation of quiescent 86.1 cells in the absence of other growth factors and serum constituents other than bovine serum albumin, transferrin, and lipids.