• Chitosan, a component of the cell wall of many fungi, has been widely used to mimic pathogen attack and has been shown to induce several defence responses. • Here we show that low concentrations (50 μ g ml^-1) of chitosan are able to induce an increase in cytosolic Ca²⁺ concentration ([ Ca²⁺] _cyt), accumulation of H₂ O₂ in the culture medium, induction of the defence gene chalcone synthase (chs), and cell death in soybean cells (Glycine max). • Chitosan-induced cell death occurred through cytoplasmic shrinkage, chromatin condensation and activation of caspase 3-like protease, suggesting the activation of a programmed cell death (PCD) pathway. Buffering extracellular Ca²⁺ with the Ca²⁺ chelator EGTA prevents [ Ca²⁺] _cyt elevation, H₂ O₂ production and all downstream PCD features, but not cell death. • Higher doses (200 M g ml^-1) of chitosan evoked neither Ca²⁺ transient and H₂ O₂ production nor caspase 3-like activation, but caused cell death, possibly as a result of plasma membrane disturbance.