Harmonic imaging is a new contrast-specific imaging modality, which utilizes the nonlinear properties of microbubble-based sonographic contrast agents by transmitting at the fundamental frequency but receiving at the second harmonic frequency. The feasibility of improving the detection of slow, small-volume blood flow using real-time harmonic imaging has been investigated in vivo. Proteinaceous microspheres (FS069) were administrated to four dogs, two woodchucks (with multiple hepatomas), and one rabbit. Three different scanners were used to obtain real-time images of kidneys and liver (including vessels) in harmonic and conventional gray scale and color flow modes. The duration of contrast enhancement lasted significantly longer in harmonic than in conventional modes (on average 87 s; P = 0.008). Harmonic images were less susceptible to artifacts, such as acoustic shadowing, and a clear increase in the (flow) signal-to-noise ratio was observed. These preliminary in vivo results demonstrate the feasibility of performing real-time, contrast-enhanced harmonic imaging, but further studies are required to establish clinical efficacy.