Early events occurring during the hypersensitive resistance response (HR) were examined using the avrRpm1/RPM1 gene-for-gene interaction in Arabidopsis challenged by Pseudomonas syringae pv. tomato. Increases in cytosolic Ca2+ were measured in whole leaves using aequorin-mediated bioluminescence. During the HR a sustained increase in Ca2+ was observed which was dependent on the presence of both a functional RPM1 gene product and delivery of the cognate avirulence gene product AvrRpm1. The sequence-unrelated avirulence gene avrB, which also interacts with RPM1, generated a significantly later but similarly prolonged increase in cytosolic Ca2+. Accumulation of H2O2 at reaction sites, as revealed by electron microscopy, occurred within the same time frame as the changes in cytosolic Ca2+. The NADPH oxidase inhibitor diphenylene iodonium chloride did not affect the calcium signature, but did block H2O2 accumulation and the HR. By contrast, the calcium-channel blocker LaCl3 suppressed the increase in cytosolic Ca2+ as well as H2O2 accumulation and the HR, placing calcium elevation upstream of the oxidative burst.