Acute superfusion of nerve growth factor (NGF; 1-100 ng/ml) through a naive rat spinal cord preparation did not alter basal or electrically evoked release of substance P-like immunoreactivity (SP-LI). In contrast, neurotrophin-3 (NT-3; 1-100 ng/ml), although not modifying SP-LI basal outflow, dose-dependently inhibited the electrically evoked, but not capsaicin (10 nM)-induced, release of the peptide. This NT-3 (10 ng/ml)-induced inhibition persisted even in the presence of 100 ng/ml NGF in the perfusion fluid and was still significant when the evoked release of SP-LI was enhanced by a prolonged in vivo treatment with NGF. Co-superfusion with naloxone (0.1 microM), but not CGP 36742 (100 microM), a GABAB antagonist, prevented NT-3 (10 ng/ml) inhibition of SP-LI release. Basal and electrically evoked release of SP-LI from the rat spinal cord in vitro was not modified 24 hr after single systemic injection of either NGF (1 mg/kg) or NT-3 (10 mg/kg). At these time intervals from administration, NGF had induced thermal and mechanical hyperalgesia in the rat hindpaw, and NT-3 had induced mechanical, but not thermal, hypoalgesia. NT-3 administered six times over a 2 week period (at 1 mg/kg) did not alter thermal threshold but significantly reduced electrically evoked release of SP-LI from the spinal cord. An identical treatment regimen with 1 mg/kg NGF induced a significant increase in evoked release of SP-LI. However, this was not associated with a significant hyperalgesia. Although finding that NGF-induced hyperalgesia does not clearly correlate with changes in the release of SP-LI in the spinal cord, this study shows that NT-3 is an inhibitor of SP-LI release and suggests that this mechanism may be responsible for NT-3-induced antinociception.