Polyamines (PAs) produce H2O2 and nitric oxide (NO) during their normal catabolism and modulate plant growth and development. To explore the biochemical basis of PAs-induced growth inhibition in Triticum aestivum L seedlings, we examined the role of O2·-, H2O2 or NO in shoot and root development. Although all PA treatments resulted in a variable reduction of root and shoot elongation, spermine (Spm) caused the greater inhibition in a similar way to that observed with the NO donor, sodium nitroprusside (SNP). In both cases, O2·- production was completely blocked whereas H2O2 formation was high in the root apex under SNP or Spm treatments. Catalase recovered root and shoot growth in SNP but not in Spm-treated plants, revealing the involvement of H2O2 in SNP-root length reduction. The addition of the NO scavenger, cPTIO, restored root length in SNP- or Spm-treated plants, respectively, and partially recovered O2·- levels, compared to the plants exposed to PAs or SNP without cPTIO. A strong correlation was observed between root growth restoration and O2·- accumulation after treating roots with SNP + aminoguanidine, a diamine oxidase inhibitor, and with SNP + 1,8-diaminoctane, a polyamine oxidase inhibitor, confirming the essential role of O2·- formation for root growth and the importance of the origin and level of H2O2. The differential modulation of wheat growth by PAs through reactive oxygen species or NO is discussed. Graphical abstract Polyamines, nitric oxide and ROS interaction in plants during plant growth.