Recreational use of 3,4-methylenedioxypyrovalerone (MDPV) in the early 2000s prompted numerous scientific investigations of its behavioral and neurochemical effects. The purpose of this study was to further characterize the interoceptive stimulus effects of MDPV using a validated in-vivo drug-detection assay. Male Sprague-Dawley rats were trained to discriminate 0.3 mg/kg MDPV from saline under a fixed ratio 20 (FR 20) schedule of food reinforcement. After stimulus control was established with MDPV (∼35 training sessions), substitution tests were commenced with drugs from several chemical classes, including drugs with predominantly dopaminergic actions [MDPV, D-amphetamine, (+)-methamphetamine, (−)-cocaine], drugs with predominantly serotonergic actions [(+)-lysergic acid diethylamide, (+)-fenfluramine], and drugs with both serotonergic and dopaminergic actions (3,4-methylenedioxymethamphetamine, 4-methylmethcathinone). Full substitution for the 0.3 mg/kg MDPV cue was observed with D-amphetamine, (+)-methamphetamine, and (−)-cocaine. Surprisingly, the 5-HT releaser (+)-fenfluramine fully substituted in half the subjects, but completely suppressed responding in the remaining subjects. 3,4-Methylenedioxymethamphetamine, 4-methylmethcathinone, and (+)-lysergic acid diethylamide failed to fully substitute for MDPV. These results indicate that the MDPV cue is similar to cues produced by drugs with predominantly dopamine-increasing effects and perhaps serotonin-releasing effects among individual subjects. Given these findings, further research is warranted to directly assess the contributions of dopamine and serotonin receptor isoforms to the discriminative stimulus functions of MDPV.