Triggering of the cell adhesion molecules L1 or N-CAM in a nerve growth cone membrane fraction from fetal rat brain with purified L1 or N-CAM or specific antibodies decreases the steady-state levels of protein tyrosine phosphorylation in the membranes. Here we report that triggering of L1 and N-CAM in the growth cone-enriched membrane fraction with a subset of antibodies directed against the extracellular region of L1 and N-CAM elicited dephosphorylation of endogenous protein substrates, indicating the presence of a cell adhesion molecule-activated phosphatase. The most prominent substrates were a membrane-associated 200-kDa protein and tubulin, both of which were dephosphorylated on tyrosine and serine/threonine residues in response to L1 or N-CAM triggering. The antibody-induced phosphatase was inhibited by agents that blocked tyrosine and serine/threonine phosphatases, including sodium orthovanadate, vanadyl sulfate, zinc cations, heparin, and sodium pyrophosphate. Purified L1 and N-CAM fragments and other antibodies reacting with the extracellular region of these adhesion molecules did not activate the phosphatase but did inhibit tyrosine phosphorylation. These properties suggested that triggering of L1 and N-CAM can lead to either phosphatase activation or tyrosine kinase inhibition in growth cone membranes. These findings implicate protein phosphatases in addition to tyrosine kinases as components of L1 and N-CAM intracellular signaling pathways in growth cones.