The possible local peripheral and spinal (intrathecal) antinociceptive effect of Na+–K+–2Cl− cotransporter (NKCC) inhibitors was investigated in the rat formalin test. Nociceptive flinching behavior induced by formalin (1%) injection in the hind paw was assessed following administration of cotransporter inhibitors. Local peripheral pretreatment in the ipsilateral paw with bumetanide (ED30, 27.1±12.7 μg/paw), piretanide (ED30, 109.2±21.6 μg/paw) or furosemide (ED30, 34.3±5.0 μg/paw), but not vehicle (DMSO 100%), produced dose-dependent antinociception in phase 2 of the test. Local bumetanide had the greatest effect (∼70% antinociception). Bumetanide also inhibited formalin-induced flinching behavior during phase 1 (ED30, 105.6±99.1 μg/paw). Spinal intrathecal pretreatment with bumetanide (ED30, 194.6±97.9 μg), piretanide (ED30, 254.4±104.9 μg) or furosemide (ED30, 32.0±6.9 μg), but not vehicle (DMSO 100%), also produced antinociception in phase 2. In this case, only intrathecal furosemide reduced flinching behavior during phase 1 (ED30, 99.4±51.4 μg) and had the maximal antinociceptive effect in phase 2 (∼65% antinociception). The opioid receptor-antagonist naloxone (2 mg/kg, s.c.) did not reverse antinociception induced by either peripheral or spinal administration of NKCC blockers. Our data suggest that the Na+–K+–2Cl− cotransporter localized in sensory neurons at intraspinal and peripheral sites is involved in formalin-induced nociception.