A series of regioisomeric analogues of 3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione]prop-2-en-1 -ol (EO9, NSC 382459) with the hydroxymethyl and hydroxypropenyl substituents situated at either the 2- or the 3-position of the indole ring were synthesized. The compound lacking the 2-hydroxypropenyl substituent (31) had similar properties to EO9 under both aerobic and hypoxic conditions against V79 cells and was more potent against a human tumour cell line (A549) than EO9. It was reduced by human DT-diaphorase (DTD) at more than double the rate of EO9, thus implicating the importance of the enzyme activation step. Compound 16 (lacking the 3-hydroxymethyl substituent) was a better substrate for human DTD than EO9, yet exhibited lesser toxicity under both aerobic and hypoxic conditions. The toxicity generated by 16 was attributed to the 5-aziridinyl moiety and suggests a greater contribution from the 3-substituent over the 2-substituent. The toxicity of EO9 was attributed to a combination of the aziridinyl group and the leaving group properties of the 3-hydroxymethyl substituent. In general, compounds with a 5-methylaziridinyl moiety, such as EO8, exhibited substantially better hypoxia-selectivity due to much slower reduction by DTD (20-fold), thus reducing aerobic potency. All compounds had similar electron affinities, as indicated by their one-electron reduction potentials.