^99mTc-ECD SPECT is valuable for the evaluation of cell viability and function. The purpose of the present study was to evaluate the significance of ^99mTc-ECD brain SPECT in ischemic stroke. We compared ^99mTc-ECD brain SPECT with perfusion and diffusion weighted images (PWI, DWI). Ten patients with acute and early subacute ischemic stroke were included in this prospective study. T2-weighted images (T2WI), DWI, PWI and ^99mTc-ECD SPECT were obtained during both the acute/early subacute and late subacute stages. In the case of PWI, time to peak (TTP) and regional cerebral blood volume (rCBV) maps were obtained. The rCBV map and ^99mTc-ECD SPECT images were compared in 8 lesions using Δ AI. The asymmetry index (AI) was calculated as (Ci - Cc) × 200 / (Ci + Cc); where Ci is the mean number of pixel counts of an ipsilateral lesion and Cc is the mean number of pixel counts of the normal contralateral hemisphere. Δ AI was defined as AI_acute - AI_subacute in the ischemic core and periphery. PWI and ^99mTc-ECD SPECT detected new lesions of the hyperacute stage or of evolving stroke more accurately than T2WI and DWI. ^99mTc-ECD SPECT was able to localize the infarct core and peri-infarct ischemia in all lesions in both the acute and the subacute stages. Δ AI was higher in the rCBV map than in the ^99mTc-ECD SPECT images in the ischemic core (p = 0.063) and in the periphery (p = 0.091). In the ^99mTc-ECD SPECT images, Δ AI was higher in the ischemic core than in the periphery (p = 0.028). During the subacute stage, ^99mTc-ECD SPECT detected all the lesions without the pseudonormalization seen in the MR images of 5/11 lesions. Based on this study, ^99mTc-ECD SPECT is comparable to PWI in terms of its ability to detect acute stroke and is more useful than PWI in the case of subacute infarction.