Real-time ultrasound perfusion imaging (rt-UPI) allows visualization of microbubbles flowing through the cerebral microvasculature. We hypothesized that analysis of microbubble tissue replenishment would enable for characterization of perfusion deficits in acute middle cerebral artery (MCA) territory stroke. Twenty-three patients (mean age 70.2±13.2 years, 9 weeks) were included. Sequential images of bubble replenishment were acquired by transcranial rt-UPI at low mechanical index immediately after microbubble destruction. Different parameters were calculated from regions of interest (ROIs): real-time time to peak (rt-TTP), rise rate (b), and plateau (A) of acoustic intensity, and A¾b was used as an index of blood flow. Results were compared with diffusion-weighted and perfusion magnetic resonance imaging. Parameters of rt-UPI had lower values in ROIs of ischemic as compared with normal tissue (b = 0.58 ± 0.40 versus 1.25 ± 0.83; P = 0.001; A = 1.44 ± 1.75 versus 2.63 ± 2.31; P = 0.05; A¾b = 1.14 ± 2.25 versus 2.98 ± 2.70; P = 0.01). Real-time time to peak was delayed in ischemic tissue (11.43 ± 2.67 versus 8.88 ± 1.66 seconds; P < 0.001). From the analysis of receiver operating characteristic curves, b and A¾b had the largest areas under the curve with optimal cutoff values of b < 0.76 and A¾b < 1.91. We conclude that rt-UPI with analysis of microbubble replenishment correctly identifies ischemic brain tissue in acute MCA stroke.