Background: In human and veterinary medicine, monophasic action potential (MAP) analysis and determination of local refractory periods by contact electrode technique gives valuable information about local cardiac electrophysiological properties. It is used to investigate dysrhythmias and the impact of drugs on the myocardium. Precise measurement of total MAP duration is difficult, therefore the MAP duration is usually determined at a repolarization level of 90% (APD90). Until now, no studies are published about the feasibility of this technique in the standing non-sedated horse. In 6 healthy Warmblood horses, on two different days, an 8F quadripolar contact catheter was passed through a jugular introducer sheath and placed under ultrasound guidance at the level of the intervenous tubercle or right atrial free wall (RA), and in the right ventricular apex (RV) to record the MAP. The MAP amplitude and APD90 were measured at a resting sinus rhythm (heart rate of 30-42 bpm) and at pacing cycle lengths (PCL) of 1000 and 600 ms. The effective refractory period (ERP) was determined at PCL of 1000 and 600 ms. Results: The overall mean (+/- SD) APD90 (rest), APD90 (1000) and APD90 (600) were 263 +/- 39 ms, 262 +/- 41 ms, 236 +/- 47 ms for the RA and 467 +/- 23 ms, 412 +/- 38 ms, 322 +/- 29 ms for the RV. The mean ERP1000 and ERP600 were 273 +/- 24 ms and 256 +/- 22 ms for the RA and 386 +/- 40 ms and 293 +/- 30 ms for the RV. The measurement variability for the amplitude, APD90 and ERP measurements in the RA ranged between 36 and 44, 9-22 and 7-8%, respectively. The measurement variability for the amplitude, APD90 and ERP measurements in the RV ranged between 49 and 66, 6-7 and 10-12%, respectively. Conclusions: RA and RV MAP duration and ERP can be obtained by a contact electrode in standing non-sedated horses. The measurement variability varies with catheter location.