Artificial nitric oxide (NO) donors are widely used as tools to study the role of NO in plants. However, reliable and reproducible characterisation of metabolic responses induced by different NO donors is complicated by the variability of their NO release characteristics. The latter are affected by different physical and biological factors including temperature and light. Here we critically evaluate NO release characteristics of the donors sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO) and nitric oxide synthase (NOS), both in vitro and in planta (Nicotiana tabacum L. cv. BelW3) and assess their effects on NO dependent processes such as the transcriptional regulation of the mitochondrial alternative oxidase gene (AOX1a), accumulation of H(2)O(2) and induction of cell death. We demonstrate that, contrary to NOS and SNP, GSNO is not an efficient NO generator in leaf tissue. Furthermore, spectrophotometric measurement of NO with a haemoglobin assay, rather than diaminofluorescein (DAF-FM) based detection, is best suited for the quantification of tissue NO. In spite of the different NO release signatures by SNP and NOS in tissue, the NO dependent responses examined were similar, suggesting that there is a critical threshold for the NO response.