Subharmonic imaging (SHI) improves the isolation of ultrasound contrast agent signals from the surrounding tissue signals by transmitting at f 0 and receiving at f0/2. In this study we investigated the feasibility of generating real-time 3D (i.e., 4D) SHI volumes. Four-dimensional SHI (f0 = 5.8 MHz, transmitting 4 cycle pulses) was implemented on a Logiq 9 ultrasound scanner with a mechanically controlled 4D10L probe (GE Healthcare, Milwaukee, WI). Experimental software provided access to both individual slice data and rendered images. This software also allowed imaging in B-mode (at 10 MHz) and harmonic imaging (HI; f0 = 5 MHz, f receive= 10 MHz). Two canines received 5 μl/kg bolus injections of Definity (Lantheus Medical Imaging, N Billerica, MA) for HI and 20 μl/kg bolus for SHI of the lower renal pole. The contrast-to-tissue ratio was then calculated for three injections in each canine and compared between modes. Patients scheduled for biopsy of a mammographically identified breast lesion provided informed consent. Baseline scanning was performed in both B-mode and power Doppler. Patients then received a 0.25 ml bolus of Definity for HI followed by a bolus injection of 20 μl/kg for SHI. Baseline 2D cine loops, 3D slice data and rendered volumes were saved for off-line comparison. While scan volumes varied, a volume of 2.5 cm × 2.5 cm × 2.5 cm acquired at a 19°volume angle resulted in 45-50 slices per volume at an acquisition rate of 1.8 to 2.2 Hz depending on depth. In the canine model, SHI resulted in significant improvement in contrast visualization and rendered SHI images demonstrated superior tissue suppression and a greater overall view of renal vasculature. In breast lesions, enhancement varied greatly on a case by case basis. However, SHI resulted in improved tissue suppression and detection of contrast in almost all cases. Future work will explore the ability of 4D SHI to classify breast lesions.