Objective: The purpose of this study was to examine the feasibility of M-mode tissue Doppler imaging for localizing the accessory pathway in patients with Wolff-Parkinson-White (WPW) syndrome. Methods: Two-dimensional guided tissue Doppler M-mode was recorded at the mitral and tricuspid annular levels in 13 WPW patients. Time intervals were measured from the onset of the δ wave or the R wave to the beginning of the ventricular systolic motion. The earliest contraction site was defined as the site demonstrating the shortest time interval, and compared with the earliest activated site determined by body surface mapping and the successful ablation site. Results: In 6 patients with a left-sided pathway, tissue Doppler localization was identical to the ablation site. In 3 with a left-sided pathway and 3 with a right-sided pathway, localization was judged as an adjacent region of the ablation site. In 1 patient with a right lateral pathway, the pathway location was misdiagnosed. The tissue Doppler diagnosis for the left-sided pathways correlated well with the ablation site, in contrast to the right-sided pathways (p = 0.05). Prediction of the accessory pathway localization by tissue Doppler M-mode was equivalent to localization based on body surface mapping. Conclusions: In WPW syndrome, tissue Doppler M-mode can detect the earliest contraction sites and seems helpful in localizing the left-sided accessory pathways, but is of limited use for right-sided pathways.