Transparent microelectrodes can enable electrophysiology with simultaneous optical stimulation or imaging directly at the electrode site. Owing to their high surface area, chemical and mechanical stability and biocompatibility, carbon nanomaterials are promising candidates for such electrodes, but the challenge of balancing transparency and impedance must be solved. In this work, carbon nanostructures were deposited on transparent substrates by low temperature thermal chemical vapor deposition. Scanning electron microscopy (SEM) and Raman spectroscopy confirmed the presence of carbon. The nanostructures were deposited on 30 µm-diameter microelectrodes with indium tin oxide leads. Optical transmittance measurements demonstrated up to 40% transparency of carbon layers. Impedance spectroscopy confirmed impedance values in line with standard opaque microelectrodes. Electrophysiology with cultured chicken ventricular myocytes using these semitransparent electrodes matched conventional microelectrode array performance with signals up to 3 mV and noise of 20 µV peak-to-peak. Ultrastructure of the cell–electrode interface revealed by focused ion beam cross-sectioning and SEM imaging showed stability of the carbon electrodes after cell culture. Altogether, a robust fabrication procedure for semitransparent carbon electrodes was demonstrated, and first results showed their suitability for simultaneous electrophysiology and optical investigation. top