DNAzymes are catalytic DNA molecules capable of cleaving RNA substrates and therefore constitute a possible gene-suppression technology. We examined whether the previously reported potency of a DNAzyme targeting c-jun (Dz13) could be improved with judicious use of sequence and chemical modifications. Catalytic activity was measured to establish correlations between catalytic activity and biological potency. Surprisingly, Dz13 had significant cytotoxic activity against cells of rodent origin (IC50 = 20-50 nM) despite having greatly reduced catalytic activity against a rodent target substrate (<25%), the latter being the result of a mismatch to the rodent c-jun sequence. In contrast, a modified Dz13 matching the rodent c-jun sequence (DT1501b) had no activity at similar concentrations against human or rodent cells despite being able to efficiently cleave the rodent c-jun sequence. Overall, catalytic activity against synthetic substrates did not correlate with cytotoxic activity and catalytically inactive mutants had in some cases equal or superior potency in cell cytotoxicity assays. Further examination of other previously published DNAzymes (Rs6 and DzF) revealed other occurrences of this anomalous behaviour. The active sequences all have G-rich 5′ termini, suggesting that G-quadruplex formation might be involved. Consistent with this, deaza-guanosine substitutions abrogated cytotoxicity of Dz13. However, Dz13 did not show evidence of quadruplex formation as determined by circular dichroism studies and native electrophoresis. These data reveal that the biologic activity of several published DNAzymes is not mediated through the catalytic degradation of target mRNA.