Total and viable cell counts, differential mitotic cell counts, and incorporation of tritiated thymidine were used to study the kinetics of suspension cultures of HeLa cells exposed to urea concentrations of 0.5-1.5%. Aside from any nonspecific osmotic effects, urea concentrations of 1.0-1.4% exhibited significant cytokinetic and cytotoxic effects. Most characteristically, mitotic cells arrested in metaphase began to accumulate 4-6 hours after addition of urea and reached a peak at 15-18 hours. Thus when the cells were in the S-phase or at the S/G2 boundary at the time of addition of urea, they exhibited metaphase arrest. Subsequently, cultures continuously exposed to urea showed a decline in the mitotic index, indicating that the entry rate of cells into mitosis is lower than the rate at which cells escape from the mitotic block. Such cultures exhibited numerous abnormal and abortive mitoses and a decrease in growth and viability of the cell populations. In contrast to the initial single wave of arrested mitosis seen with continuous exposure to urea, intermittent exposure on alternate days resulted in successive waves of arrested metaphases and had considerably more pronounced effects on the growth and viability of the cultures.