In this study, we have addressed the issue of neural circuit formation using the mouse spinal cord as a model system. Our primary objective was to assess the suitability of organotypic cultures from embryonic mouse spinal cord to investigate, during critical periods of spinal network formation, the role of the local spinal cellular environment in promoting circuit development and refinement. These cultures offer the great advantage over other in vitro systems, of preserving the basic cytoarchitecture and the dorsal-ventral orientation of the spinal segment from which they are derived [Eur J Neurosci 14 (2001) 903; Eur J Neurosci 16 (2002) 2123]. Long-term embryonic spinal cultures were developed and analyzed at sequential times in vitro, namely after 1, 2, and 3 weeks. Spatial and temporal regulation of neuronal and non-neuronal markers was investigated by immunocytochemical and Western blotting analysis using antibodies against: a) the non-phosphorylated epitope of neurofilament H (SMI32 antibody); b) the enzyme choline acetyltransferase, to localize motoneurons and cholinergic interneurons; c) the enzyme glutamic acid decarboxylase 67, to identify GABAergic interneurons; d) human eag-related gene (HERG) K(+) channels, which appear to be involved in early stages of neuronal and muscle development; e) glial fibrillary acidic protein, to identify mature astrocytes; f) myelin basic protein, to identify the onset of myelination by oligodendrocytes. To examine the development of muscle acetylcholine receptors clusters in vitro, we incubated live cultures with tetramethylrhodamine isothiocyanate-labeled alpha-bungarotoxin, and we subsequently immunostained them with SMI32 or with anti-myosin antibodies. Our results indicate that the developmental pattern of expression of these markers in organotypic cultures shows close similarities to the one observed in vivo. Therefore, spinal organotypic cultures provide a useful in vitro model system to study several aspects of neurogenesis, gliogenesis, muscle innervation, and synaptogenesis.