The effects of spirohydantoin mustard (SHM), a potential antitumor agent for central nervous system tumors, in the in vitro 9L rat brain tumor model were studied. In cell culture medium at 37 degrees, the drug was totally detoxified within 30 min. Dose-response curves for exponentially growing and plateau-phase cells were similar and indicated that a small fraction of cells were resistant to SHM. When exponentially growing cells were treated with SHM (5 microgram/ml for 1 hr), recovery from potentially lethal damage occurred within 28 hr. When cells were perturbed by SHM, the S, G2, and M phases were prolonged, there was a G2 block, some cells entered mitosis, but few divided, and cells tended to accumulate in mid-S, then moved synchronously to G2-M. The rate at which cells moved was concentration dependent and was much slower at high concentrations. The ability of SHM to both synchronize cells and block DNA synthesis may be useful in multiagent therapy regimens.