To determine the domains of the neural cell adhesion molecule L1 involved in neurite outgrowth, we have generated monoclonal antibodies against L1 and investigated their effects on neurite outgrowth of small cerebellar neurons in culture. When the 10 antibodies were coated as substrate, only antibody 557.B6, which recognizes an epitope represented by a synthetic peptide comprising amino acids 818 to 832 at the border between the fibronectin type III homologous repeats 2 and 3, was as efficacious as L1 in promoting neurite outgrowth, increasing intracellular levels of Ca2+, and stimulating the turnover of inositol phosphates. These findings suggest that neurite outgrowth and changes in these second messengers are correlated. Such a correlation was confirmed by the ability of Ca2+ channel antagonists and pertussis toxin to inhibit neurite outgrowth on L1 and antibody 557.B6. These observations indicate for the first time a distinct site on cell surface-bound L1 as a prominent signal-transducing domain through which the recognition events appear to be funneled to trigger neurite outgrowth, increase turnover of inositol phosphates, and elevate intracellular levels of Ca2+.