Background: In an accompanying paper (Asmuth et al.) it was found necessary to include cell death explicitly to estimate parameters of cell proliferation. The use of bivariate flow cytometry to estimate the phase durations and the doubling times of cells labeled with thymidine analogues is well established. However, these methods of analysis do not consider the possibility of cell death. This report demonstrates that estimating cell death in G 2 /M is possible. Methods: Mathematical models for the experimental quantities, the fraction of labeled undivided cells, the fraction of labeled divided cells, and the relative movement were developed. These models include the possibility that, of the cells with G 2 /M DNA content, only a certain fraction will divide, with the remainder dying after some time T R . Simulation studies were conducted to test the possibility of using simple methods to estimate phase durations and cell death rates. Results: Cell death alters the estimates of phase transit times in a rather complex manner that depends on the lifetime of the doomed cells. However, it is still possible to obtain estimates of the phase durations of cells in S and G 2 /M and the death rates of cells in G 2 /M. Conclusions: The methods presented herein provide a new way to characterize cell populations that includes cell death rates and common measurements of cell proliferation. q 2005 Wiley-Liss, Inc.Key terms: cell cycle; phase durations; population growth; bromodeoxyuridineThe identification of DNA-synthesizing cells through the use of halogenated analogues of thymidine, such as bromodeoxyuridine, is a well-established procedure (1-6). Beyond merely measuring the S-phase fraction, the combination of DNA and halogenated thymidine analogue bivariate cytometry permits the more challenging measurement of dynamic quantities, including phase durations of S (T S )and G 2 /M (T G2M ) and a measurement of the doubling time of the population, the potential doubling time (T pot ) (7-21). These quantities have proved to be useful in a variety of applications including extensive analyses of clinical data (22)(23)(24)(25)(26)(27)(28)(29).However, the effects of cell death on these estimates have largely been ignored. In the accompanying paper it was observed that cell loss was critical in assessing the progression of T lymphocytes infected with human immunodeficiency virus (30). Moreover, the biological significance of T pot , a measurement of overall cell proliferation, is somewhat obscure. As defined operationally by Steel (31), T pot is a measurement of the difference between the observed doubling time of a cell population and that predicted by the duration of DNA synthesis time and the S-phase fraction. In the absence of evidence to the contrary, cell death was assumed to be random, with all cells in a population being equally likely to die. As explored by , violation of the random death assumption leads to very different values for T pot . In this paper, the effects of cell death in G 2 /M are considered and a method for ...