A series of substituted 2-(4-methoxyphenyl)-1H-benzimidazoles were synthesized and evaluated as inhibitors of topoisomerase I. The presence of a 5-formyl-, 5-(aminocarbonyl)-, or 5-nitro group (i.e., substituents capable of acting as hydrogen bond acceptors) correlated with the potential of select derivatives to inhibit topoisomerase I. In contrast to bi- and terbenzimidazoles, the substituted benzimidazoles that were active as topoisomerase I poisons exhibited minimum or no DNA binding affinity. 5-Nitro-2-(4-methoxyphenyl)-1H-benzimidazole exhibited the highest activity and was significantly more active than the 4-nitro positional isomer. The 5- and 6-nitro derivatives of 2-(4-methoxyphenyl) benzoxazole, 2-(4-methoxyphenyl)benzothiazole, and 2-(4-methoxyphenyl)indole were synthesized and their relative activity as topoisomerase I inhibitors determined. None of these heterocyclic analogues were effective in significantly inhibiting cleavable-complex formation in the presence of DNA and topoisomerase I, suggesting a high degree of structural specificity associated with the interaction of these substituted benzimidazoles with the enzyme or the enzyme-DNA complex. In evaluating their cytotoxicity, these new topoisomerase I poisons also exhibited no significant cross-resistance against cell lines that express camptothecin-resistant topoisomerase I.