Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.