The shape of dose-response curves obtained for asynchronous, exponentially growing 9L rat brain tumor cells treated in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea changed as a function of the drug exposure time. For short treatment times (less than 1 hr), the dose-response curves had shoulders, indicating that the cells may accumulate sublethal damage; however, after longer treatments (greater than 1 hr), little if any shoulder was apparent. The slope of the exponential portion of the dose-response curve increased progressively with treatment periods from 15 min to 2 hr. Longer exposure times (up to 24 hr) produced no further changes in the cell-kill kinetics. Cell survival was directly related to the BCNU exposure dose [o integral t Co(t)dt] and to the amount of bound BCNU per cell. Extrapolation of the curves for these two variables indicated that some BCNU damage accumulates before death occurs. The amount of serum and cell products available in the medium to bind BCNU affected the level of survival; however, there was no evidence that extracellular spontaneous breakdown products or chemical transformation products were involved in the cell-killing mechanism.