The possible participation of the nitric oxide (NO)-cyclic GMP-protein kinase G (PKG)-K⁺ channel pathway on melatonin-induced local antinociception was assessed during the second phase of the formalin test. The local peripheral ipsilateral, but not contralateral, administration of melatonin (150—600 μg/paw) produced a dose-related antinociception during both phases of the formalin test in rats. Moreover, local pretreatment with N^G-l-nitro-arginine methyl ester (l-NAME, NO synthesis inhibitor, 10–100 μg/paw), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, guanylyl cyclase inhibitor, 5–50 μg/paw), (9S, 10R, 12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo [1,2,3-fg:3′,2′,1′-kl]pyrrolo [3,4-i][1,6] benzodiazocine-10-carboxylic acid methyl ester (KT-5823, specific PKG inhibitor, 50–500 ng/paw), glibenclamide (ATP-sensitive K⁺ channel blocker, 5–50 μg/paw), apamin (small-conductance Ca²⁺-activated K⁺ channel blocker, 0.1–1 μg/paw) or charybdotoxin (large- and intermediate-conductance Ca²⁺-activated K⁺ channel blocker, 0.03–0.3 μg/paw), but not N^G-d-nitro-arginine methyl ester (d-NAME, inactive isomer of l-NAME, 100 μg/paw) or vehicle, significantly prevented melatonin (300 μg/paw)-induced antinociception. Data suggest that melatonin-induced local peripheral antinociception during the second phase of the test could be due to activation of the NO-cyclic GMP-PKG-ATP-sensitive and Ca²⁺-activated K⁺ channels pathway.