Accurate three dimensional (3D) mapping of bioelectric sources in the body with high spatial resolution is important for the diagnosis and treatment of a variety of cardiac and neurological disorders. Ultrasound current source density imaging (UCSDI) is a new technique that maps electrical current flow in tissue. UCSDI is based on the acousto-electric (AE) effect, an interaction between electrical current and acoustic pressure waves propagating through a conducting material and has distinct advantages over conventional electrophysiology (i.e., without ultrasound). In this study, UCSDI was used to simultaneously image current flow induced in two tissue phantoms positioned at different depths. Software to simulate AE signal was developed in Matlab™ to complement the experimental model and further characterize the relationship between the ultrasound beam and electrical properties of the tissue. Both experimental and simulated images depended on the magnitude and direction of the current, as well as the geometry (shape and thickness) and location of the current sources in the ultrasound field (2.25MHz transducer). The AE signal was proportional to pressure and current with detection levels on the order of 1 mA/cm2 at 258kPa. We have imaged simultaneously two separate current sources in tissue slabs using UCSDI and two bridge circuits to accurately monitor current flow through each source. These results are consistent with UCSDI simulations of multiple current sources. Real-time 3D UCSD images of current flow automatically co-registered with pulse echo ultrasound potentially facilitates corrective procedures for cardiac and neural abnormalities.