Held et al. (1984a,b) demonstrated previously that glutathione (GSH), a negatively charged thiol, is significantly less efficient in the hydrogen atom donation repair reaction with radicals induced by radiation in transforming DNA (t-DNA) than are other thiol compounds. Fahey et al. (1991a,b) postulated that the charge on thiols can influence their ability to radioprotect DNA. GSH, which is excluded from the vicinity of DNA due to its negative charge, is less protective than neutral or positively charged thiols. We have investigated this phenomenon further with trypanothione, the conjugate of glutathione and spermidine, N1,N8-bis (L-gamma-glutamyl-L-hemicystinyl-glycyl)-spermidine. Trypanothione exists in aerobic solution largely as the disulphide (T(S)2) but is maintained in the cell in the reduced form (T(SH)2) by means of an NADPH-dependent flavo-enzyme, trypanothione reductase (TR). Experimental data show that T(S)2 in the presence of TR radioprotects t-DNA in the absence of oxygen much better than GSH or spermidine alone or in combination. Little radioprotection by T(S)2 is seen when TR is not present. The results obtained with reduced trypanothione at low concentrations suggest that radioprotection of t-DNA in hypoxia occurs predominantly by H atom donation and slightly by .OH radical scavenging, and the protection is greater than that by GSH or spermidine because the polyamine moiety in trypanothione allows a greater concentration of GSH near the DNA molecule.