Real-time 3-dimensional (RT3D) echocardiography has the potential to precisely identify the position of an object in 3-dimensional space. Therefore, we hypothesized that RT3D echocardiography could rapidly facilitate accurate placement of a bioptome within the right ventricle and may offer advantages over a fluoroscopically guided approach. During 63 routine right ventricular biopsy procedures (total of 315 biopsy attempts) in 33 cardiac allograft recipients, the bioptome was initially guided against the intraventricular septum with the use of biplane fluoroscopy. Bioptome position was then evaluated by RT3D echocardiography by using the Volumetrics Model 1 with the transducer placed at the apex. Multiple long-axis and short-axis planes were simultaneously visualized and customized to identify the tip of the bioptome. Bioptome placement was prospectively classified as septal, free wall/septal junction, or free wall. Of the 36 patients studied, 33 (91%) had adequate RT3D images. Of 315 bioptome placements visualized by RT3D echocardiography after fluoroscopic placement, bioptome position against the septum was confirmed in 113 (36%), against the septal/free wall (anterior or posterior) junction in 140 (44%), against the free wall in 60 (19%), and in the coronary sinus in 2 (1%). RT3D echocardiography is readily feasible for use in the majority of transplant patients undergoing right ventricular endomyocardial biopsy. Visualization of the bioptome in multiple simultaneous planes allows accurate localization of the biopsy site. The potential for improved localization of the bioptome tip in the right ventricle may have important clinical implications for augmenting the efficacy of this procedure.