Methoctramine and its analogues are polymethylene tetramines that selectively bind to a variety of receptor sites. Although these compounds are widely used as pharmacological tools for receptor characterization, the toxicological properties of these polyamine-based structures are largely unknown. We have evaluated the cytotoxic effects of methoctramine and related symmetrical analogues differing in polymethylene chain length between the inner nitrogens against a panel of cell lines. Methoctramine caused cell death only at high micromolar concentrations, whereas its pharmacological action is exerted at nanomolar level. Increasing the spacing between the inner nitrogen atoms resulted in a significative increase in cytotoxicity. In particular, an elevated cytotoxicity is associated to a methylene chain length of 12 units dividing the inner amine functions (compound 5). H9c2 cardiomyoblasts were the most sensitive cells, followed by SH-SY5Y neuroblastoma, whereas HL60 leukaemia cells were much more resistant. Methoctramine and related compounds down-regulated ornithine decarboxylase, the first enzyme of polyamine biosynthesis even at non-toxic concentration. Further, methoctramine and compound 5 caused a limited up-regulation of spermine/spermidine N-acetyltransferase, suggesting that interference in polyamine metabolism is not a primary mechanism of toxicity. Methoctramine and its analogues bound to DNA with a higher affinity than spermine, but the correlation with their toxic effect was poor. The highly toxic compound 5 killed the cells in the absence of caspase activation and caused an increase in p53 expression and ERK1/2 phosphorylation. Compound 5 was directly oxidized by cell homogenates producing hydrogen peroxide and its toxic effect was partially subdued by the inhibition of its uptake, by the NMDA ligand MK-801, and by the antioxidant N-acetylcysteine, suggesting that compound 5 can act at different cellular levels and lead to oxidative stress.