Studies in mice and rats have shown that antinociception produced by intrathecal (i.t.) administration of opioids can be partially inhibited by intracerebroventricular (i.c.v.) administration of naloxone. In this study we tested the hypothesis that this inhibition by i.c.v. naloxone results from antagonism of supraspinal endogenous opioid-mediated antinociception produced by the action of i.t. opioids on an ascending antinociceptive pathway. In rats lightly anesthetized with urethane/alpha-chloralose, i.t. DAMGO, i.t. lidocaine, or spinal transection at T5-T6 all attenuated the trigeminal jaw opening reflex (JOR) (i.e., were antinociceptive), an effect that was antagonized in each case by i.c.v. naloxone. These findings support the suggestion that there exists a pathway that ascends from the spinal cord to a supraspinal site that tonically inhibits antinociception mediated by supraspinal opioids. When activity in this ascending pathway is suppressed (e.g., by i.t. opioids or local anesthetics or by spinal cord transection), antinociception mediated by supraspinal opioids is disinhibited. To determine the supraspinal site(s) at which endogenous opioid-dependent antinociception is evoked by i.t. opioids, we microinjected naloxone methiodide into several supraspinal sites. Microinjection of naloxone methiodide into nucleus accumbens but not into the rostral ventral medulla (RVM) or the periaqueductal gray matter (PAG) antagonized the suppression of the JOR produced by i.t. DAMGO or lidocaine. The possibility that this ascending pathway may represent a source of spinal input to mesolimbic circuitry involved in setting the state of sensorimotor reactivity to noxious stimuli is discussed.