Illudins are novel low molecular weight natural products cytotoxic to human tumor cells in vitro. Illudin-derived analogs are effective against experimental human cancers nonresponsive to conventional anticancer agents. It is not known why some illudin analogs are more efficacious in vitro and in vivo than other analogs. Therefore, the in vitro cytotoxicity of the parent compound illudin S towards tumor cells was characterized using radiolabeled drug. Two cell lines sensitive at nanomolar concentrations using only a 15-min exposure period displayed a saturable, energy-dependent accumulation of illudins with relatively low K(m) and high Vmax values. A nonsensitive cell line, requiring millimolar concentrations to achieve in vitro toxicity, showed minimal illudin uptake with higher K(m) and lower Vmax values. No release of radioactivity could be demonstrated from tumor cells, indicating that there was no efflux of illudin S (or metabolites) from these cells. The number of intracellular illudin S molecules required to kill 50% of cells of different tumor cell lines varied from 78000 to 1114000 molecules per cell and was correlated with the 2-h IC50 value determined using a colony-forming assay. Illudin S was cytotoxic to a variety of multidrug-resistant tumor cell lines regardless of whether resistance was mediated by gp170/mdrl, gp180/MRP, GSHTR-pi, topoisomerase I, topoisomerase II, increased DNA repair capacity, or alterations in intracellular thiol content. Information obtained in this study could be used to design clinical phase I trials and to develop analogs with improved therapeutic indexes.