We developed new nonradioactive microspheres and used more sensitive X-ray fluorescence spectrometers than used previously to measure regional blood flow in the heart and other organs. We demonstrated the chemical stability of eight kinds of heavy element-loaded microspheres and validated their use for regional blood flow measurement by comparing duplicate flows measured with radioactive and/or nonradioactive microspheres in both acute and chronic dog experiments. The wavelength-dispersive spectrometer (Philips PW 1480) has a higher sensitivity than the previously described X-ray fluorescent system and reduced the number of microspheres required for accurate measurement. The fine energy resolution of this system makes it possible to increase the numbers of different kinds of microspheres to be quantitated, but at present only eight kinds are available. We also used a synchrotron radiation-excited energy dispersive spectrometer. The monochromatic synchrotron radiation allowed us to obtain much higher signal-to-background ratios of X-ray fluorescence spectra than with the wavelength-dispersive system (50 dB more for Zr-loaded microspheres) and will enable analysis of fluorescent activity in smaller regions (< 20 mg) than the radioactive method does.