Cultured neurons become attached to hydrogel substrates prepared from 2-hydroxyethylmethacrylate but grow few nerve fibers unless fibronectin, collagen, or nerve growth factor is incorporated into the hydrogel. Antibodies to fibronectin inhibit nerve fiber growth on hydrogels containing fibronectin, which suggests that growing neurons interact directly with proteins trapped in the hydrogel. The adhesive requirements for attachment of neurons appear distinct and possibly less specific than those for fiber growth. Defined hydrogel substrates offer a controlled method for analyzing complex substrates that support nerve fiber growth and neuronal differentiation.