Recently, thyroid 99m Tc-methoxyisobutylisonitrile ( 99m Tc-MIBI) scintiscanning has been proposed in an attempt to preoperatively identify thyroid malignancies, but discrepant results have been reported for oncocytic lesions. The aim of this study was to investigate the usefulness of visual and semiquantitative analyses of 99m Tc-MIBI scintigraphy for preoperatively characterizing thyroid nodules with indeterminate cytologic diagnoses, segregating in advance nononcocytic variants from those that are oncocytic. This study also aimed to analyze the relationship between 99m Tc-MIBI images and P-glycoprotein (P-gp)/multidrug resistance-associated protein-1 (MRP1) immunohistochemical expression. Methods: Fifty-one consecutive patients with cold thyroid nodules cytologically diagnosed as nononcocytic or oncocytic follicular neoplasm were prospectively studied. Visual and semiquantitative 99m Tc-MIBI scanning was performed and the diagnoses of the lesions were histologically proven by subsequent thyroidectomy. Immunohistochemical evaluation of P-gp and MRP1 was also performed on surgical samples. Results: Visual and semiquantitative 99m Tc-MIBI scintiscans showed a low specificity in preoperatively discriminating malignant oncocytic lesions. In nononcocytic nodules, the semiquantitative method was more accurate than the visual (94.44% and 77.78%, respectively). P-gp protein expression was negative in all thyroid lesions, whereas apical plasma membrane MRP1 expression was found in 78% of the lesions with a negative 99m Tc-MIBI retention index, compared with 11% of lesions with a positive retention index, correlating most strongly with a negative 99m Tc-MIBI RI in those cases with strong MRP1 apical expression. Conclusion: Semiquantitative 99m Tc-MIBI scintigraphy is an adjunctive method to predict preoperatively the malignant behavior of nononcocytic follicular thyroid nodules indeterminate at fine-needle aspiration biopsy, with a potential impact on the definition of their clinical management. Moreover, the good correlation found between immunohistochemical apical expression of MRP1 and the scintigraphic findings supports the 99m Tc-MIBI results and provides tissue information on the molecular mechanisms responsible for 99m Tc-MIBI images in thyroid lesions.