Activation of cardiac sympathetic afferents during myocardial ischaemia causes angina and induces important cardiovascular reflex responses. Reactive oxygen species (ROS) are important chemical stimuli of cardiac afferents during and after ischaemia. Iron‐catalysed Fenton chemistry constitutes one mechanism of production of hydroxyl radicals. Another potential source of these species is xanthine oxidase‐catalysed oxidation of purines. Polymorphonuclear leukocytes (PMNs) also contribute to the production of ROS in some conditions. The present study tested the hypothesis that both xanthine oxidase‐catalysed oxidation of purines and neutrophils provide a source of ROS sufficient to activate cardiac afferents during ischaemia. We recorded single‐unit activity of cardiac afferents innervating the ventricles recorded from the left thoracic sympathetic chain (T1‐5) of anaesthetized cats to identify the afferents' responses to ischaemia. The role of xanthine oxidase in activation of these afferents was determined by infusion of oxypurinol (10 mg kg−1, I.V.), an inhibitor of xanthine oxidase. The importance of neutrophils as a potential source of ROS in the activation of cardiac afferents during ischaemia was assessed by the infusion of a polyclonal antibody (3 mg ml−1 kg−1, I.V.) raised in rabbits immunized with cat PMNs. This antibody decreased the number of circulating PMNs and, to a smaller extent, platelets. Since previous data suggest that platelets release serotonin (5‐HT), which activates cardiac afferents through a serotonin receptor (subtype 3,5‐HT3 receptor) mechanism, before treatment with the antibody in another group, we blocked 5‐HT3 receptors on sensory nerve endings with tropisetron (300 μg kg−1, I.V.). We observed that oxypurinol significantly decreased the activity of cardiac afferents during myocardial ischaemia from 1.5 ± 0.4 to 0.8 ± 0.4 impulses s−1. Similarly, the polyclonal antibody significantly reduced the discharge frequency of ischaemically sensitive cardiac afferents from 2.5 ± 0.7 to 1.1 ± 0.4 impulses s−1. However, pre‐blockade of 5‐HT3 receptors eliminated the influence of the antibody on discharge activity of the afferents during ischaemia. This study demonstrates that ROS generated from the oxidation of purines contribute to the stimulation of ischaemically sensitive cardiac sympathetic afferents, whereas PMNs do not play a major role in this process.