TRPA1 mediates formalin-induced pain. Formalin injection into the hind paw of rodents induces a biphasic pain response. The first phase is thought to result from direct activation of primary afferent sensory neurons, while the second phase is attributed to central sensitization. This study shows that formalin activates sensory neurons by directly activating TRPA1, a cation channel involved in inflammatory pain. Formalin induced calcium influx in TRPA1-expressing cells, and these responses were blocked by a TRPA1-selective antagonist. TRPA1-deficient mice showed no formalin sensitivity. Pharmacologic or genetic ablation of TRPA1 significantly reduced formalin-evoked pain behaviors. These findings indicate that TRPA1 is the principal site of formalin's pain-producing action in vivo, and that its activation underlies the physiological and behavioral responses in the formalin model of pain hypersensitivity. The formalin model is widely used to assess pain and evaluate analgesic drugs in laboratory animals. It involves injecting a dilute formalin solution into the paw and assessing pain-related behaviors over two phases. Compounds affecting Phase I include local anesthetics, while those affecting Phase II include analgesics that inhibit only the second phase. The mechanism by which formalin triggers C-fiber activation remains unclear, but this study shows that formalin activates TRPA1 in primary afferent sensory neurons. TRPA1 is activated by irritants such as AITC and allicin, and formaldehyde, the active ingredient in formalin, resembles these compounds. Formalin activates TRPA1 in heterologous expression systems and isolated sensory neurons. Blockade of TRPA1 in vivo using an antagonist or genetic ablation significantly attenuates formalin-evoked pain. These results demonstrate that formalin elicits primary sensory neuron excitation and pain through direct activation of TRPA1. The study also shows that TRPA1 is the sole target for the specific excitatory actions of formalin on sensory neurons. Pharmacologic or genetic disruption of TRPA1 blocks formalin-evoked pain. The selective TRPA1 antagonist HC-030031 blocks both AITC- and formalin-induced currents and calcium responses. These findings support the conclusion that TRPA1 is the principal site of formalin action. The study reveals the molecular mechanism of formalin-induced pain, showing that formalin specifically activates TRPA1 and drives the firing of a subpopulation of C fibers. This activation likely parallels that of other TRPA1 agonists, which induce covalent modification of cysteine and lysine residues. Formalin-evoked TRPA1 activation drives CNS sensitization that underlies Phase II of the formalin response. The results suggest that TRPA1 is a useful target for the development of novel analgesics.TRPA1 mediates formalin-induced pain. Formalin injection into the hind paw of rodents induces a biphasic pain response. The first phase is thought to result from direct activation of primary afferent sensory neurons, while the second phase is attributed to central sensitization. This study shows that formalin activates sensory neurons by directly activating TRPA1, a cation channel involved in inflammatory pain. Formalin induced calcium influx in TRPA1-expressing cells, and these responses were blocked by a TRPA1-selective antagonist. TRPA1-deficient mice showed no formalin sensitivity. Pharmacologic or genetic ablation of TRPA1 significantly reduced formalin-evoked pain behaviors. These findings indicate that TRPA1 is the principal site of formalin's pain-producing action in vivo, and that its activation underlies the physiological and behavioral responses in the formalin model of pain hypersensitivity. The formalin model is widely used to assess pain and evaluate analgesic drugs in laboratory animals. It involves injecting a dilute formalin solution into the paw and assessing pain-related behaviors over two phases. Compounds affecting Phase I include local anesthetics, while those affecting Phase II include analgesics that inhibit only the second phase. The mechanism by which formalin triggers C-fiber activation remains unclear, but this study shows that formalin activates TRPA1 in primary afferent sensory neurons. TRPA1 is activated by irritants such as AITC and allicin, and formaldehyde, the active ingredient in formalin, resembles these compounds. Formalin activates TRPA1 in heterologous expression systems and isolated sensory neurons. Blockade of TRPA1 in vivo using an antagonist or genetic ablation significantly attenuates formalin-evoked pain. These results demonstrate that formalin elicits primary sensory neuron excitation and pain through direct activation of TRPA1. The study also shows that TRPA1 is the sole target for the specific excitatory actions of formalin on sensory neurons. Pharmacologic or genetic disruption of TRPA1 blocks formalin-evoked pain. The selective TRPA1 antagonist HC-030031 blocks both AITC- and formalin-induced currents and calcium responses. These findings support the conclusion that TRPA1 is the principal site of formalin action. The study reveals the molecular mechanism of formalin-induced pain, showing that formalin specifically activates TRPA1 and drives the firing of a subpopulation of C fibers. This activation likely parallels that of other TRPA1 agonists, which induce covalent modification of cysteine and lysine residues. Formalin-evoked TRPA1 activation drives CNS sensitization that underlies Phase II of the formalin response. The results suggest that TRPA1 is a useful target for the development of novel analgesics.