Introduction: Optical mapping of transmembrane potential (Vm) is an important tool in the investigation of impulse propagation in the heart. It provides valuable information about spatiotemporal changes of Vm that cannot be obtained by other techniques, but it presently is limited to measurements from the heart surfaces. Therefore, the goal of this work was to develop a technique for intramural multisite optical measurements of V m using fiberoptic technology. Methods and Results: An optrode, a bundle of thin optical fibers, was developed for measuring intramural optical signals at multiple sites in the heart. The optrode consisted of seven fibers with diameter of 225 μm arranged in a hexagonal pattern that were used to deliver excitation light to the myocardium, to collect the emitted fluorescence, and to project the light onto a 16 x 16 array of photodiode detectors. Rabbit hearts were stained with the Vm-sensitive dye RH-237. Fluorescence was excited using a 100-W Hg lamp. Intramural action potentials were recorded at multiple sites separated by 2 mm inside the left ventricle. Signal-to-noise (RMS) ratio was 21.2 ± 12 (n = 7) without averaging or ratiometry and with negligible cross-talk (<1.9%) between the neighboring photodiodes. The size of the recording area for an individual fiber was estimated at approximately 0.8 mm. Conclusion: These data demonstrate feasibility of multisite transmural measurements of Vm without signal averaging and ratiometry. This technique might become useful in studies of transmural impulse conduction during arrhythmias and defibrillation.