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Tc-99m MIBI can be excluded from cytosol against its concentration gradient as a suitable transport substrate by P-glycoprotein. Tc-99m MIBI has also been shown to be more effective than Tl-201 chloride for evaluating the response to chemotherapy in patients with small-cell lung cancer. The relation between Tc-99m MIBI accumulation by the tumor and its response to chemotherapy were evaluated in patients with non–small-cell lung cancer (NSCLC) and compared with the same parameters achieved using Tl-201 chloride.Thirty-eight patients with NSCLC were examined before chemotherapy was begun. They were classified according to the results of a follow-up computed tomogram into two groups: responders were patients in whom there was a ≥50% decrease and nonresponders were patients in whom there was a <50% decrease in the sum of the product of the maximum perpendicular diameters of all measurable lesions. All patients underwent dual-isotope imaging with Tl-201 chloride and Tc-99m MIBI just before chemotherapy. Regions of interest were placed over the tumor uptake (T) and contralateral normal lung tissue (N) areas on one coronal view with a clearly defined lesion, and the T:N ratio and retention index were calculated.The delayed T:N ratio and retention index for Tc-99m MIBI in the responder group were significantly greater (P < 0.05) than those in the nonresponder group. There was no significant correlation between the T:N ratio and retention index and tumor response using Tl-201 chloride.Tc-99m MIBI SPECT may be more effective than Tl-201 chloride SPECT for evaluating the response to chemotherapy in patients with NSCLC.