In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors (Y27632 and Fasudil), a cyclooxygenase-1 selective inhibitor (SC560), and a cyclooxygenase-2 inhibitor (NS398). We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance. In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors (Y27632 and Fasudil), a cyclooxygenase-1 selective inhibitor (SC560), and a cyclooxygenase-2 inhibitor (NS398) cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to stimulate neurite outgrowth induction in both cell lines, while SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.