We have recently identified a synthetic peptide, termed C3, capable of binding the first immunoglobulin-like module of neural cell adhesion molecule (NCAM) by means of combinatorial chemistry and shown that this NCAM ligand promotes neurite outgrowth. By means of single cell calcium imaging using the calcium-sensitive probe fura-2-acetomethyl ester, we here show that the C3-peptide induced an increase in intracellular calcium in primary hippocampal neurons and PC12-E2 cells, presumably requiring mobilization of calcium from both extracellular and intracellular stores. We further observed that C3-induced neurite outgrowth was inhibited by antagonists of voltage-dependent calcium channels as well as by an inhibitor of intracellular calcium mobilization, TMB-8. These findings demonstrate at the single cell level that a synthetic NCAM ligand directly can induce an increase in intracellular calcium and suggest that NCAM-dependent neurite outgrowth requires calcium mobilization from both extracellular and intracellular calcium stores. Thus, the C3-peptide may be regarded as a useful tool for the study of NCAM-dependent signal transduction. Furthermore, the peptide may be of considerable therapeutical interest for the treatment of neurodegenerative disorders.