Improved endocardial border delineation with the application of contrast agents should allow for less complex and faster tracing algorithms for left ventricular volume analysis. We developed a fast rotating phased array transducer for 3D imaging of the heart with harmonic capabilities making it suitable for contrast imaging. In this study the feasibility of 3D harmonic contrast imaging is evaluated in vitro. A commercially available tissue mimicking flow phantom was used in combination with Sonovue. Backscatter power spectra from a tissue and contrast region of interest were calculated from recorded radio frequency data. The spectra and the extracted contrast to tissue ratio from these spectra were used to optimize the excitation frequency, the pulse length and the receive filter settings of the transducer. Frequencies ranging from 1.66 to 2.35 MHz and pulse lengths of 1.5, 2 and 2.5 cycles were explored. An increase of more than 15 dB in the contrast to tissue ratio was found around the second harmonic compared with the fundamental level at an optimal excitation frequency of 1.74 MHz and a pulse length of 2.5 cycles. Using the optimal settings for 3D harmonic contrast recordings volume measurements of a left ventricular shaped agar phantom were performed. Without contrast the extracted volume data resulted in a volume error of 1.5%, with contrast an accuracy of 3.8% was achieved. The results show the feasibility of accurate volume measurements from 3D harmonic contrast images. Further investigations will include the clinical evaluation of the presented technique for improved assessment of the heart.