A dual isotope, single photon emission tomography (SPECT) technique using 99Tcm-hexamethylpropyleneamine oxime (HMPAO) and 123I-iodoamphetamine (IMP) was investigated to determine its suitability for assessing regional cerebral blood flow (rCBF) changes resulting from cognitive activation. The similarity of the 123I-IMP and 99Tcm-HMPAO distributions under the same physiological conditions was first investigated by administering the two agents to human subjects (n = 8) either simultaneously or at different times but during the performance of the same task. Normalized ratio images generated from the 99Tcm and 123I data showed that the two tracers distributed similarly in the left and right cerebral hemispheres when administered under similar physiological conditions. There was, however, a significant anterior/posterior gradient that appears to be the result of partial volume effects due to small differences in spatial resolution of the two agents. In two subjects, 99Tcm-HMPAO was administered during a resting period with eyes-closed and 123I-IMP was injected later during visual checkerboard stimulation. Ratio images showed a localized increase in the occipital lobes during the visual stimulation consistent with the expected increase in rCBF. The dual isotope strategy appears promising for study of changes in rCBF due to cognitive activation.