In order to develop a novel Tc-99m-labeled folate based SPECT radiotracer with an optimized pharmacokinetic profile for the folate receptor (FR) positive tumor imaging, a folate conjugate, HYNIC-PEG2-FA, was designed, synthesized and radiolabeled with Tc-99m using tricine/diphenylphosphinobenzene-3-sulfonic acid sodium (TPPMS), tricine/trisodium triphenylphosphine-3,3',3 ''-trisulfonate (TPPTS), and ethylenediamine-N,N'-diacetic acid (EDDA) as coligands. Tc-99m(HYNIC-PEG2-FA)(tricine/TPPTS), 4, Tc-99m(HYNIC-PEG2-FA)(tricine/TPPMS), 5 and Tc-99m(HYNIC-PEG2-FA)(EDDA), 6, were obtained respectively. All of them were stable in saline and mouse plasma for 6 h, and displayed high specific binding in the FR-positive KB cell line in vitro. Among them, complex 4 exhibited a higher tumor uptake (11.35 +/- 0.67 ID% g(-1) at 2 h p.i.) and more rapid clearance from the liver, lungs, blood, muscle and other non-target organs than Tc-99m (HYNIC-NHHN-FA)(tricine/TPPTS), which we reported before. Small animal SPECT/CT imaging studies in FR-positive KB tumor models showed that the tumor could be clearly visualized at 120 min p.i., suggesting its potential as a promising folate receptor targeting agent for tumor imaging.