The cell-membrane folic acid (FA) receptors are known to be responsible for cellular accumulation of FA and FA analogs, such as methotrexate (MTX), and are overexpressed on several tumor cells. Folate, as well as antifolates (i.e., MTX), possess high affinity for the folate-receptor positive cells and tissues and were deemed useful for diagnostic imaging. We have prepared and evaluated technetium-99m (99mTc)- labeled FA and MTX analogs using MAG3 and MAG2 chelating agents in an attempt to develop folate-receptor targeting radiopharmaceuticals. Folate and MTX-conjugates after labeling with 99mTc by ligand exchange method displayed high in vitro stability in human plasma. In vitro cell binding and internalization on MCF-7 and MDA-MB-231 cells indicated the affinity and specificity of the radioconjugates toward human breast cancer cells. In mice, all radioconjugates showed rapid clearance from the blood and excretion mainly through the renal/urinary pathway, with some elimination by way of the biliary route. There was no significant accumulation of radioactivity observed in other organs, with the exception of the intestines. Uptake in the breast tumor was moderate in nude mice. These findings could be of potential diagnostic interest in designing and developing FA/MTX-based radiopharmaceuticals for tumor imaging.