Thymosin beta(4) (Tbeta4) is a major actin-sequestering peptide widely distributed in mammalian tissues including the nervous system. The presence of this peptide in the nervous system likely plays a role in synaptogensis, axon growth, cell migration, and plastic changes in dendritic spine. However, the effects of Tbeta4 on the survival of neurons and axonal outgrowth have still not been fully understood. So far it is not clear if the effects of Tbeta4 are associated with L1 functions. In the present study, we hypothesized that Tbeta4-induced up-regulation of L1 synthesis could be involved in the survival and axon outgrowth of cultured spinal cord neurons. To test this hypothesis, primarily cultured neurons were prepared from the mouse spinal cord and treated with various concentrations of Tbeta4 ranging from 0.1 to 10 microg/ml. The analysis of L1 mRNA expression and protein synthesis in neurons was then carried out using RT-PCR and western blot assays, respectively. After the addition of Tbeta4 to cultures, cells were then treated with antibodies against distinct domains of L1-Fc. Subsequently, beta-tubulin III and L1 double-labeled indirect immunofluorescence was carried out. Meanwhile, L1 immunofluorescent reactivity was analyzed and compared in cells treated with Tbeta4. Furthermore, the number of beta-tubulin III-positive cells and neurite lengths were measured. We found that Tbeta4 enhanced L1 expression in a dose-dependent manner, and the highest L1 mRNA and protein synthesis in cells increased by more than 2.1- and 2.3-fold in the presence of Tbeta4 at identical concentrations, respectively. Moreover, it also dose dependently enhanced neurite outgrowth and neuronal survival. Compared to conditions without Tbeta4, the length of neurite and neuronal survival increased markedly in presence of 0.5, 1, and 5 microg/ml Tbeta4, respectively, whereas the effects of Tbeta4 were significantly attenuated or inhibited in the process of L1-Fc antibodies treatment. These above results indicate that the promotive effect of Tbeta4 on the survival and neurite outgrowth of cultured spinal cord neurons might be mediated, at least in part via a stimulation of the production of L1 in the neurons.