Extracellular fluid levels of glutamate were measured in the locus coeruleus during butorphanol (a mixed agonist at mu-, delta-, and kappa-opioid receptors) withdrawal by using microdialysis in conscious butorphanol-dependent Sprague-Dawley rats. Guide cannulae were implanted chronically and rats were given intracerebroventricular (i.c.v.) infusions of butorphanol (26 nmol/1 microliter/hr) or saline (1 microliter/hr) for 3 days. Microdialysis probes (2 mm tip) were inserted into the locus coeruleus 24 hr before precipitation of withdrawal by i.c.v. injection of naloxone (48 nmol/5 microliters). A separate series of rats was rendered dependent by peripheral injection of butorphanol (20 mg/kg, s.c., b.i.d.) for 5 days and naloxone (5 mg/kg, i.p.) was given to precipitate withdrawal. Single injections of butorphanol (26 nmol/5 microliters, i.c.v.) had no effect on the extracellular fluid levels of glutamate, compared to rats injected with vehicle. Behavioral evidence of withdrawal was detected following naloxone challenge in butorphanol-dependent rats (both i.c.v. and s.c. models), but not in non-dependent, vehicle-treated rats. Significant increases (P < 0.05) in levels of glutamate were noted after naloxone-precipitated withdrawal only in the butorphanol group. The glutamate levels in the locus coeruleus increased from 8.37 +/- 2.01 before, to 21.93 +/- 4.58 microM in the first 15 min sample following i.c.v. injections of 48 nmol/5 microliters naloxone and from 10.84 +/- 1.74 before, to 26.01 +/- 6.19 microM in the 15-30 min sample following i.p. injections of 5 mg/kg naloxone in the butorphanol-dependent rats, respectively. These results provide direct evidence to support the role of excitatory amino acids within the locus coeruleus in butorphanol withdrawal.