Our phantom studies indicate that the energy resolution (9.7% FWHM) of a new three-headed single-photon tomograph (PRISM-3000) separates the distribution of 99mTc from 123I for 10% asymmetric or 15% or 10% centered 99mTc windows when combined with a 10% asymmetric 123I window. This technique is now applied to the simultaneous measurement of resting rCBF and changes induced by vasodilation (1 g acetazolamide) in 10 subjects with cerebrovascular disease. Resting and vasodilated 133Xe SPECT images were obtained first. Within 48 hr, 99mTc HMPAO was given at rest, acetazolamide injected, and after 20 min either [123I] IMP or [123I] HIPDM was administered. Subjects were scanned for 99mTc and 123I simultaneously using 10% asymmetric windows. Regression analyses demonstrated a linear relationship between 133Xe SPECT and dual-isotope SPECT measurements of lesion-to-cerebellum ratios in baseline (r = 0.92), vasodilated (r = 0.86) and rest-minus-vasodilated data (r = 0.85). Technetium-99m and 123I images obtained through dual-isotope imaging are by definition in perfect anatomic registration.