We evaluated dependency of neutrophils' O₂^- production responses following thermal injury (30% body surface scald burn) on Protein tyrosine kinase (PTK)-Lyn and Mitogen-activated protein kinase (MAP)-Erk 1/2. Activation of PTK, Lyn was assessed by determining its autophosphorylation, and its ability to phosphorylate enolase using immunoprecipitation. Western blot analysis was performed to assess phosphorylation of Erk 1/2. O₂^- anion production was measured by isoluminol-enhanced luminometry. Imaging technique was employed to measure neutrophil [Ca²⁺]_i in individual cells. The results showed a marked Lyn and Erk 1/2 upregulation accompanying enhanced O₂^- production in neutrophils obtained from rats day 1 after burn injury. The treatment of rats with either PTK blocker (AG556), or Erk 1/2 blocker (AG1478) prior to subjecting them to burn injury caused complete inhibition of respective kinase activation. Both AG556 and AG1478 treatments produced an approximately 66% inhibition in neutrophil O₂^- production. Treatment of rats with a Ca²⁺ channel blocker diltiazem (DZ) produced an approximately 37% inhibition of O₂^- production without affecting Lyn or Erk 1/2 activation occurring with burn injury. Ca²⁺ mobilization in neutrophils was upregulated with burn injury but not affected by treatment of burn rats with PTK blocker AG556. Unlike the partial inhibition of burn-induced neutrophil O₂^- production by AG556, AG1478 or DZ, platelet activating factor antagonist (PAFa) treatment of burn rats produced a near complete inhibition of O₂^- production. PAFa treatment was also found to block activation of Lyn. The latter finding together with the earlier report of abrogation of Ca²⁺ signaling by PAFa, suggests that the near complete inhibition of O₂^- production by PAFa was a result of blockade of PTK as well as Ca²⁺ signaling pathways. Overall, our studies suggest that in the early phases of burn injury, the enhanced O₂^- production by neutrophils is a result of potentiation of Ca²⁺-linked and Ca²⁺-independent signaling pathways, which could be triggered by inflammatory agents such as platelet activating factor, PAF.