Abstract Background The Drosophila CNS midline cells are an excellent model system to study neuronal and glial development because of their diversity of cell types and the relative ease in identifying and studying the function of midline-expressed genes. In situ hybridization experiments generated a large dataset of midline gene expression patterns. To help synthesize these data and make them available to the scientific community, we developed a web-accessible database. Description MidExDB (Drosophila CNS Midline Gene Expression Database) is comprised of images and data from our in situ hybridization experiments that examined midline gene expression. Multiple search tools are available to allow each type of data to be viewed and compared. Descriptions of each midline cell type and their development are included as background information. Conclusion MidExDB integrates large-scale gene expression data with the ability to identify individual cell types providing the foundation for detailed genetic, molecular, and biochemical studies of CNS midline cell neuronal and glial development and function. This information has general relevance for the study of nervous system development in other organisms, and also provides insight into transcriptional regulation.