Rationale <p>Environmental cues guide animals towards resources vital for survival but can also drive maladaptive reward-seeking behaviours, as in gambling and eating disorders. While conditioned stimuli (CSs) are paired with reward delivery after reward-seeking actions, discriminative stimuli (DSs) signal reward availability independently of behaviour.</p> Objective <p>We introduce a procedure to compare CS and DS effects on reward-seeking behaviour, in the same subjects within a single session.</p> Methods <p>Female and male Sprague–Dawley rats learned to self-administer sucrose. During each session, DS+ trials signaled that lever pressing would produce sucrose paired with a CS+ , and DS- trials signaled no sucrose and a CS-. Next, in the absence of sucrose, we assessed the ability of the cues to <i>i)</i> reinforce lever pressing and <i>ii)</i> increase sucrose seeking when presented response-independently. We also assessed the effects of the mGlu_2/3 receptor agonist LY379268 and d-amphetamine on cue-induced sucrose seeking.</p> Results <p>By the end of self-administration training, lever pressing peaked during DS+ trials and dropped during DS- trials. The DS+ was a conditioned reinforcer of sucrose seeking in both sexes, whereas the CS+ was more effective in males. Response-independent presentations of the DS+ invigorated sucrose seeking in both sexes, whereas the CS+ was effective only in males. LY379268 suppressed DS+ -triggered sucrose seeking in females, with no effect in males. D-amphetamine enhanced sucrose seeking non-specifically across cue conditions in males, with no effect in females.</p> Conclusions <p>Our new trial-based procedure can be used to identify unique and similar mechanisms underlying DS and CS influences on appetitive behaviour.</p>