Interatrial septum (IAS) pacing has been proposed for the prevention of paroxysmal atrial fibrillation. IAS pacing is usually guided by fluoroscopy and P wave analysis. The authors have developed a new approach for IAS pacing using intracardiac echocardiography (ICE), and examined its effects on P wave characteristics. Cross-sectional images are acquired during pullback of the ICE transducer from the superior vena cava into the inferior vena cava by an electrocardiogram- and respiration-gated technique. The right atrium and IAS are then three-dimensionally reconstructed, and the desired pacing site is selected. After lead placement and electrical testing, another three-dimensional reconstruction is performed to verify the final lead position. The study included 14 patients. IAS pacing was achieved at seven suprafossal (SF) and seven infrafossal (IF) lead locations, all confirmed by three-dimensional imaging. IAS pacing resulted in a significant reduction of P wave duration as compared to sinus rhythm (99.7 +/- 18.7 vs 140.4 +/- 8.8 ms; P < 0.01). SF pacing was associated with a greater reduction of P wave duration than IF pacing (56.1 +/- 9.9 vs 30.2 +/- 13.6 ms; P < 0.01). P wave dispersion remained unchanged during septal pacing as compared to sinus rhythm (21.4 +/- 16.1 vs 13.5 +/- 13.9 ms; NS). Three-dimensional intracardiac echocardiography can be used to guide IAS pacing. SF pacing was associated with a greater decrease in P wave duration, suggesting that it is a preferable location to decrease interatrial conduction delay.