While mammography has high sensitivity in the detection of breast cancer, its low specificity results in a false-positive rate of roughly 80 %. Thus, a secondary tool for the characterization of breast masses would be highly beneficial. This ongoing study investigates the use of 4D harmonic and subharmonic imaging (HI and SHI) for the characterization of breast masses identified by mammography. Patients scheduled for an ultrasound guided biopsy of a mammographically-identified breast mass provided informed consent to undergo a contrast ultrasound study prior to biopsy. Scanning was performed using a modified Logiq 9 scanner with a 4D10L probe (GE Healthcare, Milwaukee, WI). Experimental software provided 4D HI (transmitting 2 cycle pulses at 5 MHz and receiving at 10 MHz) and 4D SHI (transmitting 4 cycle pulses at 5.8 MHz and receiving at 2.9 MHz). Subjects underwent 2D B-mode and power Doppler imaging (PDI), before receiving a 0.25 ml injection of the contrast agent Definity (Lantheus Medical Imaging, N. Billerica, MA) for 4D HI. After 30 minutes, subjects received a second injection of 20 μl/kg (up to 1.25 ml) for 4D SHI of the lesion. To date, 157 women have been enrolled and 149 have completed the study. Incomplete studies resulted from a failure to gain IV access (n=5), equipment malfunction (n=2), and a previously undisclosed contraindication to the contrast agent (n=1; unstable pulmonary status). 3D volume rates ranged from 1.7 Hz (for a 3.7×1.3×2.0 cm lesion) to 6.1 Hz (for a 6x4x6 mm lesion). SHI resulted in improved tissue suppression in 146 cases, equal tissue suppression in 2 cases, and inferior tissue suppression in 1 case relative to HI. Malignancy has been confirmed by pathology in 37 cases. Flow visualization (in all lesions) was evident in 82 cases on PDI, 8 cases on 4D HI, and 68 cases on 4D SHI. Thus, while the diagnostic value has yet to be determined by the radiologists, initial results indicate that 4D SHI provides improved tissue suppression and visualization of flow in breast masses.