Abstract: An ultrasound calibration phantom comprises a portion including at least one fiducial structure having a selected geometric arrangement; a portion adapted for mechanical coupling of the ultrasound calibration phantom to a transrectal ultrasound (TRUS) stepper; and a guide for at least one surgical instrument, the guide being mechanically coupled to the fiducial structure and/or to the TRUS stepper at a selected pose relative to the fiducial structure. A calibration method uses the calibration phantom and provides automatic, intraoperative calibration of ultrasound imaging systems. The invention is useful in ultrasound-guided clinical procedures.