Pharmacological differences among mu opioid drugs have been observed in in vitro and in vivo preclinical models, as well as clinically, implying that all mu opioids may not be working through the same mechanism of action. Here we demonstrate analgesic synergy between L-methadone and several mu opioid ligands. Of the compounds examined, L-methadone selectively synergizes with morphine, morphine-6beta-glucuronide, codeine, and the active metabolite of heroin, 6-acetylmorphine. Morphine synergizes only with L-methadone. In analgesic assays, D-methadone was inactive alone and did not enhance morphine analgesia when the two were given together, confirming that L-methadone was not acting through N-methyl-D-aspartate mechanisms. Both L-methadone and morphine displayed only additive effects when paired with oxymorphone, oxycodone, fentanyl, alfentanyl, or meperidine. Although it displays synergy in analgesic assays, the L-methadone/morphine combination does not exhibit synergy in the gastrointestinal transit assay. This analgesic synergy of L-methadone with selective mu opioid drugs and the differences in opioid-mediated actions suggest that these drugs may be acting via different mechanisms. These findings provide further evidence for the complexity of the pharmacology of mu opioids.