Our aim was to ascertain the relationship between the degree of Tc-99m-MIBI uptake and the level of p-glycoprotein (Pgp) expression determined by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) techniques in patients with hematologic malignancy. Methods: A total of 21 samples (19 patients) were evaluated. Two patients had repeat studies after therapy. Thirteen samples were studied at the time of initial diagnosis and 8 during relapse after therapy. After MIBI imaging, either bone marrow aspiration or peripheral blood was obtained for flow cytometric and RT-PCR analyses, Flow cytometry was performed using two different antibodies. After the injection of 555 MBq MIBI, whole-body and pelvic spot images were acquired using a dual-head gamma camera, The uptake in the bone marrow was evaluated against the background (adjacent soft tissue) by both qualitative (scoring system) and quantitative (tm/bkg ratios) analyses, Results: For flow cytometry, the limit for Pgp overexpression was set at >15% Pgp-positive mononuclear bone marrow or peripheral blood cells. There was an inverse correlation between the levels of Pgp and MIBI imaging using both the qualitative (scoring system) and quantitative (tm/bkg ratios) analyses (p = 0.022), Mean values were statistically different between Pgp + and Pgp - groups for both qualitative and quantitative analyses (p = 0.009 and 0.024, respectively). For RT-PCR, there was statistical support toward a difference in the mean values between Pgp + and Pgp - groups by qualitative analysis (p = 0.061); however, no statistical difference was found between these two groups by quantitative analysis (p = 0.179), Conclusion: Based on the strong correlation between the imaging and flow cytometry and a statistical support toward the correlation between the imaging and RT-PCR, MIBI imaging may be used for the in vivo detection of Pgp in patients with hematologic malignancy.