L1 is a neural cell adhesion molecule that has been shown to help guide nascent axons to their targets. This guidance is based on specific interactions of L1 with its binding partners and is likely to involve signaling cascades that alter cytoskeletal elements in response to these binding events. We have examined the phosphorylation of L1 and the role it may have in L1-directed neurite outgrowth. Cytosolic extracts from nerve growth factor-stimulated PC12 cells were fractionated by anion-exchange chromatography, and an activity was found that phosphorylated the cytoplasmic domain of L1. This activity was then assayed using a battery of L1-derived synthetic peptides. Based on these peptide assays and sequencing of radiolabeled L1 proteolytic fragments, the phosphorylation site was determined to be Ser1152. Western blot analysis demonstrated that the L1 kinase activity from PC12 cells that phosphorylated this site was co-eluted with the S6 kinase, p90(rsk). Moreover, S6 kinase activity and p90(rsk) immunoreactivity co-immunoprecipitate with L1 from brain, and metabolic labeling studies have demonstrated that Ser1152 is phosphorylated in vivo in the developing rat brain. The phosphorylation site is located in a region of high conservation between mammalian L1 sequences as well as L1-related molecules in vertebrates from fish to birds. We performed studies to investigate the functional significance of this phosphorylation. Neurons were loaded with peptides that encompass the phosphorylation site, as well as the flanking regions, and their effects on neurite outgrowth were observed. The peptides, which include Ser1152, inhibit neurite outgrowth on L1 but not on a control substrate, laminin. A nonphosphorylatable peptide carrying a Ser to Ala mutation did not affect neurite outgrowth on either substrate. These data demonstrate that the membrane-proximal 15 amino acids of the cytoplasmic domain of L1 are important for neurite outgrowth on L1, and the interactions it mediates may be regulated by phosphorylation of Ser1152.