Lesioned central nervous system (CNS) axons fail to regenerate because of limited availability of neurotrophic factors (NTF) to promote neuron survival and drive axon regeneration through an environment rich in multiple myelin- and non myelin-derived axon growth inhibitory ligands that initiate growth cone collapse through the Rho/Rho kinase (ROCK) signalling pathway. However, pharmacological inhibition of Rho and ROCK promotes neurite outgrowth in PC12, Ntera-2 cells and embryonic/early postnatal neurons in culture. We have used our well-characterised CNS myelin-inhibited adult rat retinal culture model to show that Y27632 only promotes disinhibited neurite outgrowth if RGC are co-stimulated with ciliary neurotrophic factor (CNTF). Y27632 in CNTF-stimulated retinal cultures promotes optimal RGC neurite outgrowth at 10 muM concentrations, while higher concentrations negatively correlate with RGC neurite outgrowth and survival. Raising the levels of cAMP in Y27632-treated retinal cultures also promotes significant RGC neurite outgrowth, an effect that is potentiated by the further inclusion of CNTF. Our results suggest that Y27632-induced ROCK inhibition promotes robust disinhibited axon regeneration of adult neurons only when growth promoting factors are added and/or cAMP levels are raised.