Neurite outgrowth of PC12 cells in the presence of nerve growth factor and the spreading of 3T3 fibroblasts were inhibited by human myelin proteins from different areas of the central nervous system (CNS) in a dose-dependent manner. Application of liposomes containing human CNS myelin proteins induced rapid collapse of PC12 growth cones. When 3T3 fibroblasts were plated on a human CNS myelin protein-coated substrate the cells remained round, and spreading was inhibited. All these inhibitory effects could be neutralized by the monoclonal antibody IN-1, which was raised against a 250 kDa neurite growth-inhibiting protein (NI-250) of rat CNS myelin. Comparison of the inhibitory properties of human and bovine CNS myelin on PC12 neurite outgrowth showed that human CNS myelin was slightly more inhibitory per unit of myelin protein. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that in human myelin, as in rat and bovine myelin, a high molecular weight protein is responsible for the inhibitory activities on neurite outgrowth and fibroblast spreading.