This study investigated sequence-specific cell uptake characteristics of Tat basic domain and related permeation peptides with an emphasis on residue chirality, length, and modified side chains. Effects on cell permeation of defined basic domain sequences within a library of 42 different peptides were evaluated using transport of radiolabeled peptides into human Jurkat leukemia cells. All other factors being equal, when the chirality of the peptide sequence was changed from L to D, uptake values increased up to 13-fold. Control experiments showed that the quantitative difference in uptake could not be attributed to increased decomposition of an L- versus a D-peptide by cellular or serum proteases. Furthermore, length, sequence, and type of chelation domain impacted peptide uptake into cells. The highest level of uptake was found with the following peptides: (23) D-Tat-Orn [Ac-rkkrr-orn-rrr-AHAkgc-amide] and (33) D-poly-Arg9 [Ac-rrrrrrrrr-AHA-kgc-amide]. The best of these peptide sequences could be employed as in vivo imaging and drug delivery agents to translocate substrates into cells.