Endogenous and exogenous thiols are thought to influence cellular radiosensitivity directly by radical scavenging and/or hydrogen donation processes, and indirectly, by regulating the amount of oxygen (or other electron affinic radiosensitiser) able to reach the radiosensitive targets of the cell. The relative importance of these two mechanisms was evaluated in multicell spheroids treated with two agents currently undergoing clinical testing, the thiophosphate WR-2721 and the glutathione synthesis inhibitor BSO. Fluorescence-activated cell sorting techniques were used to recover cells selectively from different depths (different oxygenation status) within the spheroids. The radiosensitivity of cell populations recovered from different regions suggested that both agents acted primarily by affecting the oxygenation status of the spheroid. Similarly, the binding of a fluorescent marker for hypoxic cells, the nitrofuran AF-2, was markedly enhanced by WR-2721 addition, and decreased by BSO-induced thiol depletion. We conclude that the major radiobiological consequence of thiol manipulation in multicell systems is to increase or decrease the availability of oxygen.