An X-ray fluorescence system with low Compton background and high counting efficiency was developed to measure regional blood flow with nonradioactive microspheres. The performance of the system was tested in vitro by counting mixed aqueous solutions of either Mo, Ag, and I; Nb, Ag, and Ba; or Zr, Mo, Rh, Ag, Sn, I, and Ba, as well as a mixture of Ag and Ba nonradioactive microspheres. Mixtures containing 2-20 ppm of each element were counted for 10 min by the fluorescence system, and the individual elements in mixtures of three to seven nonradioactive elements were measured with high accuracy. The best counting statistics were obtained for Ag. For 10-min counts, the system measures as few as 120 Ag microspheres with 30% standard deviation but measures 800 Ag microspheres per sample with 3.6% standard deviation. We compared regional myocardial blood flows determined simultaneously by fluorescence and radioactive microsphere methods; the latter samples were counted by a 3-in. NaI (Tl) well detector and pulse-height analyzer. The radioactive and nonradioactive measurements showed good correlations.