Most clinically used opioids are thought to induce analgesia through activation of the mu opioid receptor (MOR). However, disparities have been observed between the efficacy of opioids in activating the MOR
in vitro
and in inducing analgesia
in vivo
. In addition, some clinically used opioids do not produce cross-tolerance with each other, and desensitization produced
in vitro
does not match tolerance produced
in vivo
. These disparities suggest that some opioids could be acting through other targets
in vivo
, but this has not been comprehensively tested. We thus screened 9 clinically relevant opioids (buprenorphine, hydrocodone, hydromorphone, morphine, O-desmethyl-tramadol, oxycodone, oxymorphone, tapentadol, tramadol) against 9 pain-related receptor targets (MOR, delta opioid receptor [DOR], kappa opioid receptor [KOR], nociceptin receptor [NOP], cannabinoid receptor type 1 [CB1], sigma-1 receptor [σ1R], and the monoamine transporters [NET/SERT/DAT]) expressed in cells using radioligand binding and functional activity assays. We found several novel interactions, including monoamine transporter activation by buprenorphine and σ1R binding by hydrocodone and tapentadol. Tail flick anti-nociception experiments with CD-1 mice demonstrated that the monoamine transporter inhibitor duloxetine selectively promoted buprenorphine anti-nociception while producing no effects by itself or in combination with the most MOR-selective drug oxymorphone, providing evidence that these novel interactions could be relevant
in vivo
. Our findings provide a comprehensive picture of the receptor interaction profiles of clinically relevant opioids, which has not previously been performed. Our findings also suggest novel receptor interactions for future investigation that could explain some of the disparities observed between opioid performance
in vitro
and
in vivo
.