Components of the extracellular matrix (ECM) have been implicated in the regulation of neuronal migration, axonal growth, and synaptogenesis. We have examined cultures of glial cells, Schwann cells, and schwannomas for the expression of two components of the ECM, laminin and s-laminin, using immunohistochemical and Western blot techniques. Laminin is a potent promotor of neurite outgrowth in cultures of both central and peripheral neurons, and is present in all ECMs. In contrast, s-laminin (for synaptic laminin), a recently described homolog of laminin, is highly localized at the neuromuscular synaptic cleft (Sanes and Chiu, Cold Spring Harbor Symp. Quant. Biol. 1983;48:667-678; Chiu and Sanes, Dev. Biol. 1984;103:456-467) and shows selective adhesivity for motor neurons (Hunter et al. Cell 1989;59:905-913). While the distribution of these ECM components have been well documented in situ, the sources of these extracellular molecules are unclear. We report that astrocytes cultured in serum-free medium maintain an organized ECM that only bears laminin immunoreactivity; s-laminin appears to be sequestered intracellularly. However, both molecules are found in the astrocyte conditioned medium. Thus, under these growth conditions, astrocytes produce and release laminin and s-laminin, but only incorporate the former into an ECM. In contrast, neither molecule is present in comparable cultures of oligodendrocytes. Although no established ECM is seen in cultures of Schwann cells or schwannomas, laminin and s-laminin immunoreactivity are present within cells and in the conditioned media. These results indicate that certain populations of non-neuronal support cells and cell lines can produce and release both synaptic and extrasynaptic components of the ECM. The assembly of these different molecules into an organized basal lamina may require the presence of additional factors or interaction with neurons.