Encounters of regenerating goldfish retinal axons with oligodendrocytes and CNS myelin of mammals and fish were monitored in in vitro assays. Upon contact with highly branched rat oligodendrocytes, goldfish axons collapsed or grew around but never crossed these cells. However, in the presence of the antibody IN-1 against the oligodendrocyte-associated growth-inhibitory proteins, axons did grow over highly branched oligodencrocytes. In contrast to the mammalian oligodendrocytes, goldfish optic nerve/tract-derived oligodendrocytelike cells allowed the growth of axons across their surface and even along their processes. The fish growth cones avoided entering the region of rat CNS myelin applied to polylysine/laminin-coated coverslips or failed to elongate on this substrate. They were, however, able to pass over CNS myelin of fish. When exposed to rat CNS myelin as the sole substrate, axonal outgrowth from fish retinal explants was inhibited almost entirely. However, outgrowth on fish CNS myelin was substantial, but many more axons extended on fish or rat brain membranes that were depleted of myelin. Thus, goldfish retinal axons are sensitive to the axon-growth-inhibiting cell-surface molecules of mammalian oligodendrocytes as well as CNS myelin. Fish optic nerve oligodendrocytelike cells and fish CNS myelin lack these inhibitory properties and are growth permissive. These in vitro experiments suggest that the success of axonal regeneration in the fish optic nerve is causally related to the presence of growth-permissive properties and to the absence of growth inhibitors on fish optic nerve/tract oligodendrocytelike cells.