In a prospective study, we correlated the washout rate of Tc-99m-sestamibi (MIBI) and the degree of MIBI accumulation with the expression of P-glycoprotein (Pgp) in tumor tissues in a total of 46 patients with lung cancer. Methods: All patients underwent early (30 min) and delayed (3 hr) MIBI imaging and bronchoscopic biopsy before initiation of chemo- or radiotherapy. The interval between biopsy and imaging varied between 2 and 10 days. All patients had radiologically detectable tumors. Immunohistochemical studies were performed on paraffin sections using a monoclonal antibody, JSB-1, developed against the internal epitope of Pgp. Normal tissue and tumor washout rates and tumor-to-background ratios were correlated with the level of Pgp expression. Results: There was an inverse correlation between tumor-to-background ratios and the density of Pgp (p = 0.001), whereas there was no appreciable correlation between tumor washout rates of MIBI and the level of Pgp expression (p = 0.414). Conclusion: The current data strongly suggest that, although the reduced ability for the tumors to accumulate MIBI correlates well with the increased levels of Pgp expression, tumor washout rates of MIBI do not correlate with the density of Pgp in tumor tissues. Our results also warrant additional research for correlating immunohistological and imaging findings with messenger RNA levels determined by polymerase chain reaction and flow cytometry.