Clinical μ-opioid receptor (MOR) agonists produce hyperalgesic priming, a form of maladaptive nociceptor neuroplasticity, resulting in pain chronification. We have established an in vitro model of opioid-induced hyperalgesic priming (OIHP), in male rats, to identify nociceptor populations involved and its maintenance mechanisms. OIHP was induced in vivo by systemic administration of fentanyl and confirmed by prolongation of prostaglandin E2 (PGE2) hyperalgesia. Intrathecal cordycepin, which reverses Type I priming, or the combination of Src and MAP kinase (MAPK) inhibitors, which reverses Type II priming, both partially attenuated OIHP. Parallel in vitro experiments were performed on small-diameter (<30 µm) dorsal root ganglion (DRG) neurons, cultured from fentanyl-primed rats, and rats with OIHP treated with agents that reverse Type I or Type II priming. Enhancement of the sensitizing effect of a low concentration of PGE2 (10 nm), another characteristic feature of priming, measured as reduction in action ...