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High-frequency (HF) ultrasound imaging has been shown to be useful for imaging anatomical structures of the eye and small animals in biological and pharmaceutical research, achieving good spatial resolution at an affordable price. Cardiovascular research utilizing mice requires not only real-time B-scan imaging, but also ultrasound Doppler to evaluate both movements and blood flow of the mouse heart. In this paper, we report the development of the first real-time duplex HF ultrasound system with both B-scan imaging and Doppler imaging, using a 30 MHz 64-element linear array. The system included a HF pulsed-wave Doppler module, a 16-channel HF analog beamformer module, a PC with a 200 MS/s 14-bit PCI A/D card, and real-time Labview software. Both a wire phantom and a micro flow phantom were used to evaluate system performance. The system has a lateral resolution better than 160 urn and is capable of measuring motion velocity as low as 0.1 mm/s and as high as 1 m/s. B-scan images of excised rabbit eyes have been achieved, as well as clear blood flow velocity profiles in mouse superficial vessels with diameters of 200 mum and major aortas. The system is able to acquire real-time B-mode and Doppler images. The system will also have the capability of acquiring 400 B-mode images per second. In vivo zebrafish and mouse experiment results show a promising future of this system in small animal research.