Open microfluidic channels were used to separate the extracellular space around a cardiomyocyte into three compartments: the cell ends and a central partition (insulating gap). The microchannels were filled with buffer solution and overlaid with paraffin oil, thus forming the cavities for the cell ends. The central part of the cardiomyocyte rested on the partition between two adjacent microchannels and was entirely surrounded by the paraffin oil. This arrangement increased the extracellular electrical resistance to > 20 MOmega and facilitated the recording of the time course of the change in extracellular voltage and current during subthreshold and suprathreshold stimuli. The waveform of the extracellular current and voltage in response to an extracellular depolarizing stimulus comprised an initial monophasic signal followed by a biphasic signal with a delay of 2-15 ms. The latter was associated with a transient contraction and therefore caused by an action potential. The biphasic signal became monophasic after the depolarization of one cell end by raised extracellular [K+]. This form of differential recording revealed the repolarization phase of the action potential. At rest, the sarcomere length within the gap was 12% +/- 4.8% longer than outside the gap, but intracellular Ca2+ transients occurred to the same extent as that observed in the outer pools. This data demonstrate the feasibility of the use of a microfluidic bath design to limit the extracellular resistance between two ends of an isolated cardiomyocyte.