Under sufficient acoustic excitation microbubble-based ultrasound contrast agents (UCA) produce a marked subharmonic (f0/2) frequency component. By selectively receiving at the subharmonic, it is possible to perform UCA specific subharmonic imaging (SHI). We investigated the ability to quantify tissue perfusion (in ml/min/g) in 5 canines using 3D SHI. A Logiq 9 scanner (GE Healthcare, Milwaukee, WI) equipped with a 4D10L probe was modified to perform 3D harmonic imaging (HI; ftransmit=5 MHz and freceive=10 MHz) and 3D SHI (ftransmit=5.8 MHz and freceive=2.9 MHz). Imaging was performed in the lower renal pole of open-abdomen canines after injection of Definity (Lantheus Medical Imaging, N Billerica, MA). The canines received a 5 μl/kg bolus injection of Definity for HI and a 20 μl/kg for SHI in triplicate for each kidney. A microvascular staining technique based on isotope-labeled microspheres was used to quantify the degree of perfusion in each kidney. Ligation of a surgically exposed branch of the renal arteries induced low perfusion. Time-intensity curves were generated from regions-of-interest (ROIs) corresponding to the tissue samples used for microvascular staining. Perfusion was estimated from the initial slope of the fractional blood volume uptake and compared to the reference standard using linear regression analysis. Microsphere perfusion data showed an average perfusion of 9.30±6.60 and 5.15±3.42 ml/min/g pre and post ligation, respectively. The reference standard showed significant correlation with 3D HI perfusion estimates (r=0.38; p=0.007), but correlated better with 3D SHI (r=0.62; p<0.001). Additionally, these results showed an improvement 2D SHI perfusion estimates previously reported by our group (r=0.57; Forsberg et al JUM 2006). In conclusion, 3D SHI perfusion estimates were in better overall agreement with the reference standard than 3D HI and were superior to previously reported 2D SHI results.