N-type calcium channels modulate the release of key pro-nociceptive neurotransmitters such as glutamate and substance P (SP) in the central nervous system. Considerable research interest has focused on the therapeutic potential of the peptidic ω-conopeptides, GVIA and MVIIA as novel analgesic agents, due to their potent inhibition of N-type calcium channels. Recently, the novel peptidic N-type calcium channel blocker, AM336, was isolated from the venom of the cone snail, Conus catus. Thus, the aims of this study were to (i) document the antinociceptive effects of AM336 (also known as CVID) relative to MVIIA following intrathecal (it) bolus dosing in rats with adjuvant-induced chronic inflammatory pain of the right hindpaw and to (ii) quantify the inhibitory effects of AM336 relative to MVIIA on K+-evoked SP release from slices of rat spinal cord. Both AM336 and MVIIA inhibited the K+-evoked release of the pro-nociceptive neurotransmitter, SP, from rat spinal cord slices in a concentration-dependent manner (EC 50 values= 21.1 and 62.9 nM, respectively), consistent with the antinociceptive actions of ω-conopeptides. Following acute it dosing, AM336 evoked dose-dependent antinociception (ED 50≈ 0.110 nmol) but the doses required to produce side-effects were an order of magnitude larger than the doses required to produce antinociception. For it doses of MVIIA≤ 0.07 nmol, dose-dependent antinociception was also produced (ED 50≈ 0.016 nmol). Unexpectedly, however, it doses of MVIIA> 0.07 nmol, produced a dose-dependent decrease in antinociception but the incidence and severity of the side-effects continued to increase for all doses of MVIIA investigated, suggesting that dose-titration with MVIIA in the clinical setting, may be difficult.