The efficacy of mafosfamide purging depends on factors like incubation time, drug and erythrocyte concentration. To determine the influence of protein-bound SH groups in the incubation medium, the cytotoxicity of mafosfamide on G-CSF mobilized CD34+/- cells was evaluated by short-term culture assays and drug concentration measurements. 100 micromol/ml mafosfamide was incubated for 30 min in five buffers (PBS, PBS with 1%, 5% and 10% BSA and plasma). The mean calculated areas under the concentration-time curves (AUC) were 2489 +/- 198, 1561 +/- 286, 976 +/- 201, 585 +/- 62 and 605 +/- 196 micromol/l/min. The mean reductions of CFU-GM growth were 79.4%, 73.0%, 62.5%, 30.3%, 6.2% respectively. Similar results were obtained for BFU-E. Regression analysis showed a good correlation between cytotoxicity and AUCs (CFU-GM: r = 0.8195; BFU-E: r = 0.8207). This effect is well explained by the different concentrations of SH moieties in the incubation medium resulting in a higher drug binding capacity. The profound difference between AUCs and CFU-GMs in plasma and 10% BSA cannot be explained by the quantity of SH-groups. It is probably due to an additional enzymatic drug degeneration by the 3'-5'exonuclease subsite of plasma DNA polymerase. In conclusion, the concentration of albumin-associated SH groups strongly influences the cytotoxicity of mafosfamide. It has to be considered as a new and important aspect in ex vivo bone marrow purging.