The role of platelet-activating factor (PAF) in Ca²⁺ signaling and Ca²⁺-related enhancement of reactive oxygen intermediate (ROI) generation in neutrophils of burn-injured rats was ascertained by evaluating the effect of treatment of the rats with a PAF receptor antagonist. The treatment of rats with the antagonist also allowed us to evaluate the role of PAF in the priming of neutrophil ROI response with burn in vivo. A full skin thickness burn injury was produced in anesthetized rats by exposing 30% of total body surface area to 98°C water for 10 s. Sham and burn rats were killed 1 day later, and their blood was collected to obtain neutrophils. Fluorescence-activated cell sorter analysis was used to quantify ROI production by the neutrophils. Cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) imaging technique was employed to measure neutrophil [Ca²⁺]_i in individual cells and microfluorometry for the assessment of [Ca²⁺]_i responses in suspensions of neutrophils. There was an overt enhancement of ROI generation by burn rat neutrophils. ROI release was accompanied by a marked elevation of [Ca²⁺]_i signaling. The treatment of rats with PAF receptor antagonist before burn prevented the upregulation of both [Ca²⁺]_i and ROI generation in neutrophils. These studies indicate that enhanced ROI production in neutrophils in the early stages after burn injury results from a PAF-mediated priming of the [Ca²⁺]_i signaling pathways in vivo.