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Tissue characterization would be facilitated by improved methods of calibration with which to make absolute measurements of the backscatter and attenuation of tissue or contrast agents. In this paper, the use of polystyrene microspheres is examined as a potential broadband in vitro calibration tool by employing an experimental system designed to characterize ultrasonic contrast agents. The frequency-dependent attenuation coefficient and backscatter coefficient were measured for a series of microsphere size distributions with a broadband ultrasound system using a custom-built specimen chamber and a novel suspension mixing technique. The measurements were used in a broadband reduction method to yield the backscatter coefficient. The range of ka spanned in this study is from ka=0.5 to ka=12. The broadband nature of the pulses permitted simultaneous measurement of different regimes of scattering of the microspheres (specifically, the transition region from Rayleigh to short-wavelength asymptotic scattering). Experimental results were compared with corresponding theoretical predictions for the attenuation and backscatter of solid elastic spheres in liquid and yielded good quantitative agreement. Discussion of some possible applications of this technique to the fields of tissue characterization and contrast is presented. ©1997 Acoustical Society of America.