Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system. However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties. Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium (SM 20K) guarantees acquisition of all properties known from 30 years of work on cell type-specific differentiation in this medium. Through subsequent shift to newly developed active Schneider's medium (SM active), neurons adopt additional mature properties like the ability to carry out plastic morphological changes, neurotransmitter expression and electrical activity. We introduce long-term FM-dye measurements as a tool for Drosophila primary cell cultures demonstrating the presence of increased, action potential-dependent synaptic activity in SM active. This is confirmed by patch-clamp recordings, which in addition show that SM active-cultured neurons display different spiking patterns. Furthermore, we demonstrate that transmission can be evoked in SM active cultures, revealing the existence of synaptic plasticity. Thus, these culture conditions support developmental, structural and physiological properties known or expected from the nervous system, enhancing possibilities for future experiments complementing or substituting those in nervous systems of Drosophila. © 2004 Elsevier Inc. All rights reserved.