Myocardial ischemia activates cardiac spinal afferents that mediate chest pain and excitatory reflex cardiovascular responses. Platelets are activated during myocardial ischemia and release 5-hydroxytryptamine, which stimulates abdominal spinal afferents. This study investigated the role of activated platelets in excitation of cardiac spinal afferents during ischemia. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were obtained from cats and incubated with collagen (2 mg/ml) or thrombin (5 U/ml). We observed reduction of platelets in PRP indicative of platelet activation by collagen and thrombin, respectively. Activity of single-unit, ischemia-sensitive cardiac spinal afferents was recorded from the left sympathetic chain in anesthetized cats. Injection of 1.5 ml PRP + collagen (activated platelets) into the left atrium (LA) stimulated 12 of 13 cardiac afferents. PRP + saline (nonactivated platelets, LA) and PPP + collagen did not alter activity of these afferents. PRP + thrombin (1.5 ml, LA) stimulated eight of nine other cardiac afferents, whereas PPP + thrombin did not stimulate any of the nine afferents. Antiplatelet immune serum (1 ml/kg iv) significantly decreased circulating platelets as well as neutrophils (polymorphonuclear leukocytes, PMNs) in eight other cats, and in each animal, attenuated the ischemia-related increase in activity of cardiac afferents. Conversely, responses of five separate cardiac afferents to ischemia were not diminished after treatment with anti-PMN immune serum when concentration of circulating platelets was maintained by infusion of donated PRP despite the decrease in circulating PMNs. These data indicate activated platelets stimulate ischemia-sensitive cardiac spinal afferents and contribute to activation of these afferents during ischemia.