Neurons growing on microelectrode arrays (MEAs) are promising tools to investigate principal neuronal network mechanisms and network responses to pharmaceutical substances. However, broad application of these tools, e.g. in pharmaceutical substance screening, requires neuronal cells that provide stable activity on MEAs. Cryopreserved cortical neurons (CCx) from embryonic rats were cultured on MEAs and their immunocytochemical and electrophysiological properties were compared with acutely dissociated neurons (Cx). Both cell types formed neuritic networks and expressed the neuron-specific markers microtubule associated protein 2, synaptophysin, neurofilament and gamma-aminobutyric acid (GABA). Spontaneous spike activity (SSA) was recorded after 9 up to 74 days in vitro (DIV) in CCx and from 5 to 30 DIV in Cx, respectively. Cx and CCx exhibited synchronized burst activity with similar spiking characteristics. Tetrodotoxin (TTX) abolished the SSA of both cell types reversibly. In CCx SSA-inhibition occurred with an IC50 of 1.1 nM for TTX, 161 microM for magnesium, 18 microM for D,L-2-amino-5-phosphonovaleric acid (APV) and 1 microM for GABA. CCx cells were easy to handle and developed long living, stable and active neuronal networks on MEAs with similar characteristics as Cx. Thus, these neurochips seem to be suitable for studying neuronal network properties and screening in pharmaceutical research.