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We have developed and evaluated a high-frequency, real-time pulsed Doppler and physiological signal acquisition and analysis system specifically for use in mice. The system was designed to provide sampling rates up to 125 kilosamples/s (ksps) with software controlled data acquisition and analysis in real-time. Complex fast Fourier transforms are performed every 0.1 ms (or longer up to 10 ms) to provide 0.1-ms time resolution and using 64-1024 sample segments of the Doppler audio signals resulting in frequency resolution ranging from 122-1953 Hz. The system was evaluated by its response to frequency swept signals with slopes (accelerations) and magnitudes (velocities) comparable to actual blood velocity signals in mice. Signals up to a maximum frequency of 125 kHz and a maximum acceleration of 20 MHz/s were processed and displayed. This corresponds to a maximum velocity of 480 (960) cm/s and a maximum acceleration of 750 (1500) m/s2 when Doppler shifts are measured with a 20- (10-) MHz probe, thereby allowing us to measure high stenotic jet velocities. The directional transitions of the spectrogram across zero frequency and across Nyquist frequency (sampling rate/2) were smooth with no discernible artifacts. Signals with period as low as 2 ms were processed and displayed at sweep speed that is ten times that in clinical Doppler systems, so that measurements of small temporal events can be made with precision. Thus, the new system can measure higher blood velocities with higher spatial and temporal resolution than is possible using clinical Doppler systems adapted for use in mice.