BACKGROUND: Associative learning undergirds the development of addiction, such that drug-related cues serve as conditioned stimuli to elicit drug-seeking responses. Plausibly, among opioid misusing chronic pain patients, pain-related information may serve as a conditioned stimulus to magnify opioid cue-elicited autonomic and craving responses through a process of second-order conditioning. METHODS: We utilized a novel psychophysiological probe of pain-opioid conditioned associations, the Cue-Primed Reactivity (CPR) task. In this task, participants were presented with images as primes (200鈥痬s) and cues (6000鈥痬s) in pairs organized in four task blocks: "control-opioid," "pain-opioid," "control-pain," and "opioid-pain." Opioid-treated chronic pain patients (N鈥?鈥?0) recruited from an Army base in the Western United States were classified as opioid misusers (n鈥?鈥?7) or non-misusers (n鈥?鈥?3) via a validated cutpoint on the Prescription Drug Use Questionnaire (PDUQ; Compton et al., 2008). Opioid misuse status was examined as a predictor of HRV, craving, and mood responses on the CPR task. RESULTS: HRV increased to a greater extent during the pain-opioid block compared to the control-opioid block for non-misusers compared to misusers (p鈥?鈥?003, eta(2)(partial)鈥?鈥?.27). In contrast, craving increased to a greater extent from baseline to the pain-opioid block for misusers than for non-misusers (p鈥?鈥?03, eta(2)(partial)鈥?鈥?16). CONCLUSIONS: Findings suggest that opioid-treated chronic pain patients exhibit Pavlovian conditioned responses to opioid cues strengthened by an associative learning process of second-order conditioning when primed by pain-related images. This pain-opioid contingency appears to become disrupted among individuals who engage in opioid misuse, such that opioid-related stimuli elicit motivational responses irrespective of pain-related contextual stimuli.