This study reports the composition of a chemically defined medium (DDM1) that supports the survival and differentiation of neurons in dissociated cell cultures prepared from midgastrula stage Drosophila embryos. Cells with neuronal morphology that stain with a neural-specific marker are clearly differentiated by 1 day in vitro and can be maintained in culture for up to 2 weeks. Although the whole cell capacitance measurements from neurons grown in DDM1 were 5- to 10-fold larger than those of neurons grown in a conventional serum-supplemented medium, the potassium current densities were similar in the two growth conditions. A small but significant increase in the sodium current density was observed in the neurons grown in DDM1 compared with those in serum-supplemented medium. The majority of neurons grown in DDM1 fired either single or trains of action potentials in response to injection of depolarizing current. Contributing to the observed heterogeneity in the firing properties between individual neurons grown in DDM1 was heterogeneity in the levels of expression and gating properties of voltage-dependent sodium, calcium, and potassium currents. The ability of embryonic Drosophila neurons to differentiate in a chemically defined medium and the fact that they are amenable to both voltage-clamp and current-clamp analysis makes this system well suited to studies aimed at understanding the mechanisms regulating expression of ion channels involved in electrical excitability.