We have previously reported that chicken embryos injected with a single dose of methadone (Meth) on day 3, 7 or 11 of embryogenesis fail to show dependence on day 14, measured as a significant overshoot in motility above baseline after challenge with the opioid antagonist naloxone (Nx). Constant infusion of Meth from day 7 to 14 also failed to produce evidence of dependence on day 14. To address the question of whether the 14-day-old embryo is capable of expressing withdrawal, isobutylmethylxanthine (IBMX), a compound that produces quasi-opioid withdrawal, was injected directly into the embryo, resulting in a significant increase in motility. To determine whether the 14-day-old embryo could also express true opioid withdrawal, the embryos were injected with various doses of Meth or morphine (Morph), followed at different time intervals by injections of varying doses of Nx. A high dose of Morph followed 24 hours later by a low dose of Nx produced evidence of withdrawal, as did a low dose of Meth followed 1 hour later by a higher dose of Nx, U50488H, a selective kappa agonist, had no effect on motility in the 14-day-old embryo, suggesting that the decrease in motility seen after Meth was not mediated by a kappa receptor. Pretreatment with the irreversible mu antagonist, beta-funaltrexamine (B-FNA), blocked the decrease in motility seen after Meth and also prevented the overshoot in motility when Nx was given 1 hour post-Meth. We were also able to demonstrate dependence/withdrawal in the 12-day-old embryo, but higher doses of both Meth and Nx were required.(ABSTRACT TRUNCATED AT 250 WORDS)