Salicylic acid (SA) is a phytohormone best known for its role in plant defense. It is synthesized in response to diverse pathogens and responsible for the large scale transcriptional induction of defense-related genes and the establishment of systemic acquired resistance. Surprisingly, given its importance in plant defense, an understanding of the underlying enzymology is lacking. In Arabidopsis thaliana, the pathogen-induced accumulation of SA requires isochorismate synthase (AtICS1). Here, we show that AtICS1 is a plastid-localized, stromal protein using chloroplast import assays and immunolocalization. AtICS1 acts as a monofunctional isochorismate synthase (ICS), catalyzing the conversion of chorismate to isochorismate (IC) in a reaction that operates near equilibrium (K(eq) = 0.89). It does not convert chorismate directly to SA (via an IC intermediate) as does Yersinia enterocolitica Irp9. Using an irreversible coupled spectrophotometric assay, we found that AtICS1 exhibits an apparent K(m) of 41.5 mum and k(cat) = 38.7 min(-1) for chorismate. This affinity for chorismate would allow it to successfully compete with other pathogen-induced, chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate its activity is not regulated by light-dependent changes in stromal pH, Mg(2+), or redox and that it is remarkably active at 4 degrees C consistent with a role for SA in cold-tolerant growth. Finally, our analyses support plastidic synthesis of stress-induced SA with the requirement for one or more additional enzymes responsible for the conversion of IC to SA, because non-enzymatic conversion of IC to SA under physiological conditions was negligible.