We have developed four Tc-99m(CO)(3)-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. Tc-99m(CO)(3)-hexadecylamino-N,N'-diacetic acid (negatively charged), Tc-99m(CO)(3)-hexadecylamino-N-alpha-picolyl-N'-acetic acid (uncharged), Tc-99m(CO)(3)-N,N'-dipicolylhexadecylamine (positively charged), Tc-99m(CO)(3)-N-hexadecylaminoethyl-N'-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with Tc-99m-d,l-HMPAO and [F-18]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for Tc-99m(CO)(3)-N-hexadecylaminoethyl-N'-aminoethylamine after 60 min incubation (compared to 55% and 23% for Tc-99m-d,l-HMPAO and [F-18]FDG, respectively) while in plasma-rich medium, Tc-99m(CO)(3)-N,N'-dipicolylhexadecylamine was best bound (54%, similar to the binding of 99mTc-d, l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent (Tc-99m(CO)(3)-N,N'-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent Tc-99m-d,l-HMPAO, Tc-99m(CO)(3)-N,N'-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent. (C) 2009 Elsevier Ltd. All rights reserved.