The objective of this study was to develop a kit formulation for [(TcN)-Tc-99m(mpo)(PNP5)](+) (MPO = 2-mercaptopyridine oxide), ((TcN)-Tc-99m-MPO) to support its clinical evaluations as a SPECT radiotracer. Radiolabeling studies were performed using three different formulations (two-vial formulation and single-vial formulations with/without SnCl2) to explore the factors influencing radiochemical purity (RCP) of (TcN)-Tc-99m-MPO. We found that the most important factor affecting the RCP of (TcN)-Tc-99m-MPO was the purity of PNP5. (TcN)-Tc-99m-MPO was prepared >98% RCP (n = 20) using the two-vial formulation. For single-vial formulations with/without SnCl2, beta-cyclodextrin (beta-CD) is particularly useful as a stabilizer for PNP5. The RCP of (TcN)-Tc-99m-MPO was 95-98% using beta-CD, but its RCP was only 90-93% with gamma-cyclodextrin (gamma-CD). It seems that PNP5 fits better into the inner cavity of beta-CD, which forms more stable inclusion complex than gamma-CD in the single-vial formulations. The results from biodistribution and imaging studies in Sprague-Dawley rats clearly demonstrated biological equivalence of three different formulations. Single photon-emission computed tomography data suggested that high quality images could be obtained at 0-30-min post-injection without significant interference from the liver radioactivity. Considering the ease for Tc-99m-labeling and high RCP of (TcN)-Tc-99m-MPO, the non-SnCl2 single-vial formulation is an attractive choice for future clinical studies.