BACKGROUND: The accuracy of three-dimensional mapping systems is affected by cardiac contraction and respiration. OBJECTIVE: The study sought to determine relative motion of cardiac and thoracic structures to assess positional errors and guide the choice of an optimized spatial reference. METHODS: Motion of catheters placed at the coronary sinus (CS), pulmonary vein (PV) ostia, left atrial (LA) isthmus and roof, cavotricuspid isthmus (CTI), and right atrial appendage (RAA) were recorded for 30 patients using Ensite-NavX. The right subclavian vein, left brachiocephalic vein, azygos vein, pulmonary arteries, and a static reference were included. The displacement from a mean position was calculated for each pair of sites. Respiration effects were assessed by the shift of the motion curve during in- and expiration phases. RESULTS: The PVs showed a mean interpair displacement of 4.1 +/- 0.2 mm and a shift of 5.0 +/- 0.5 mm. Proximal CS references for all LA structures (4.0 +/- 1.1 mm) were superior to the static reference (4.9 +/- 0.7 mm; P = .01). In addition, the shift due to respiration was less pronounced at 3.5 +/- 0.8 mm versus 4.9 +/- 0.5 mm (P = .004), respectively. Motion of extracardiac vessels was influenced by a mean shift of 6.8 +/- 1 mm. The remote subclavian and brachiocephalic veins were more affected (7.6 +/- 0.7 mm) than the pulmonary arteries (5.9 +/- 0.4 mm; P = .002). For the CTI, a minimized mean displacement of less than 4.6 +/- 2.0 mm relative to the proximal CS, RAA, and azygos vein was found. CONCLUSION: Respiration is the major source of relative motion, which increases with distance from the heart. For LA procedures, a proximal CS reference position is superior to a static reference position.