Rationale <p>Polysubstance use, defined as the consumption of more than one addictive substance either simultaneously or sequentially, is an established contributing factor to drug overdose deaths. Almost 80% of opioid-related overdose deaths involve a second substance and 21.6% of these deaths involve cocaine. Beyond increased overdose risk, polysubstance use is also linked to poorer treatment outcomes as well as higher relapse and mortality rates compared to single substance use. Moreover, there are no Food and Drug Administration-approved treatments for polysubstance use disorder.</p> Objectives <p>This study determined the effectiveness of two kappa opioid receptor (KOR) agonists, nalfurafine (NLF) and VZTK35, to attenuate fentanyl and cocaine co-use using a fentanyl/cocaine-vs.-food choice procedure in male and female Sprague Dawley rats.</p> Methods <p>We characterized fentanyl/cocaine mixture interactions through dose-addition analysis, cost manipulations, and repeated naltrexone treatment and examined the effects of repeated NLF and VZTK35 treatment on fentanyl-, cocaine- and fentanyl/cocaine mixture choice.</p> Results <p>Fentanyl and cocaine interactions on drug choice were synergistic and sensitive to cost manipulations but not repeated naltrexone treatment. Repeated NLF and VZTK35 treatments failed to attenuate fentanyl-, cocaine- or fentanyl/cocaine choice up to doses that disrupted operant behavior with effects of both compounds antagonized by nor-binaltorphimine.</p> Conclusions <p>While candidate medications are needed for fentanyl and cocaine co-use, these results do not support the further evaluation of these KOR agonists for this purpose. However, these methods provide an empirical preclinical framework for studying fentanyl and cocaine interactions on reinforcement endpoints.</p>