Several molecules inhibit axonal growth cones and may account for the failure of central nervous system regeneration, including myelin proteins and various chondroitan sulfate proteoglycans expressed at the site of injury. Axonal growth inhibition by myelin and chondroitan sulfate proteoglycans may in part be controlled by Rho-GTPase, which mediates growth cone collapse. Here, we tested in vitro whether pharmacological inhibition of a major downstream effector of Rho, Rho-kinase, promotes axonal outgrowth from dorsal root ganglia grown on aggrecan. Aggrecan substrates stimulated Rho activity and were inhibitory to axonal growth. Y-27632 treatment promoted the growth of axons by 5- to 10-fold and induced "steamlined" growth cones with longer filopodia and smaller lamellipodia. Interestingly, more actin bundles reminiscent of stress fibers in the central domain of the growth cone were observed when grown on aggrecan compared to laminin. In addition, Y-27632 significantly promoted axonal growth on both myelin and adult rat spinal cord cryosections. Our data suggest that suppression of Rho-kinase activity may enhance axonal regeneration in the central nervous system.