The development of a high-density active microelectrode array for in vitro electrophysiology is reported. Based on the Active Pixel Sensor (APS) concept, the array integrates 4096 gold microelectrodes (electrode separation 20 μm) on a surface of 2.5 mm x 2.5 mm as well as a high-speed random addressing logic allowing the sequential selection of the measuring pixels. Following the electrical characterization in a phosphate solution, the functional evaluation has been carried out by recording the spontaneous electrical activity of neonatal rat cardiomyocytes. Signals with amplitudes from 130 μV p-p to 300 μVp-p could be recorded from different pixels. The results demonstrate the suitability of the APS concept for developing a new generation of high-resolution extracellular recording devices for in vitro electrophysiology. © 2004 Elsevier B.V. All rights reserved.