Fasciculation and defasciculation of axons are major morphogenetic events in the formation of neuronal pathways during development. We have identified the extracellular matrix glycoprotein tenascin-R (TN-R) and its neuronal receptor, the immunoglobulin superfamily recognition molecule F3, as promoters of neurite defasciculation in cerebellar explant cultures. Perturbation of the interaction between these two molecules using both antibodies and an antisense oligonucleotide resulted in increased neurite fasciculation. The domains involved in defasciculation were identified as the N-terminal region of TN-R containing the cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats and the immunoglobulin-like domains of F3. Fasciculation induced by antibodies and the antisense oligonucleotide could be reverted by a phorbol ester activator of protein kinase C, whereas the protein kinase inhibitor staurosporine increased fasciculation. Our observations indicate that defasciculated neurite outgrowth does not only depend on the reduction of the expression of fasciculation enhancing adhesion molecules, such as L1 and the neural cell adhesion molecule (NCAM), but also on recognition molecules that actively induce defasciculation by triggering second messenger systems.