Systemic acquired resistance (SAR) is an inducible defence mechanism which plays a central role in protecting plants from microbial pathogen attack. Guided by bioassays, a new chemical inducer of SAR was isolated from the extracts of Strobilanthes cusia and identified to be 3-acetonyl-3-hydroxyoxindole (AHO), a derivative of isatin. Tobacco plants treated with AHO exhibited enhanced resistance to tobacco mosaic virus (TMV) and to the fungal pathogen Erysiphe cichoracearum (powdery mildew), accompanied by increased levels of pathogenesis-related gene 1 (PR-1) expression, salicylic acid (SA) accumulation and phenylalanine ammonia-lyase activity. To study the mode of action of AHO, its ability to induce PR-1 expression and TMV resistance in nahG transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of SA, was analysed. AHO treatment did not induce TMV resistance or PR-1 expression in nahG transgenic plants, suggesting that AHO acts upstream of SA in the SAR signalling pathway. In addition, using two-dimensional gel electrophoresis combined with mass spectrometry, five AHO-induced plant proteins were identified which were homologous to the effector proteins with which SA interacts. Our data suggest that AHO may represent a novel class of inducer that stimulates SA-mediated defence responses.