Solid tumors contain populations of proliferating (P) and quiescent (Q) cells. Shifting between these populations occurs continuously and cells are recruited from quiescence to proliferate (Q-->P) as a result of exogenously applied or endogenous cell depleting stimuli. Direct measurements of the proliferation kinetics of these Q-->P cells in solid tumors are difficult to make because of the much larger percentage of P-cells. In order to specifically analyze the kinetics of the Q-->P cells, double thymidine analogue labeling was used. This was accomplished by first labeling in vivo all of the P-cells in MCaK tumors using continuous exposure to chlorodeoxyuridine (CldUrd) administered by a minipump over 21 h. About 75% of the aneuploid cells are P-cells based on CldUrd labeling. At different times after the pumps were removed, the tumors were pulse-labeled with iododeoxyuridine (IdUrd) and harvested 6 h later. A 3-color flow cytometry assay was used to simultaneously and independently analyze CldUrd and IdUrd incorporation, as well as DNA content. The Q-->P cells were identified as having only been labeled with IdUrd. The length of their S-phase was calculated from the movement of the Q-->P cells during the 6 h after IdUrd labeling. The results showed the length of S-phase for the recruited cells to be slightly, but significantly, longer than the length of S-phase for the total cells (11 h versus 9 h, respectively). Thus, the recruited cells appear to have slightly slower kinetics than the proliferating cells in the absence of a perturbing stimulus such as radiotherapy or chemotherapy.