RANTES (CCL5) is a chemokine that recruits immune cells to inflammatory sites by interacting with the G-protein coupled receptor CCR5, which is also the primary coreceptor used together with CD4 by HIV to enter and infect target cells. Ligands of CCR5, including chemokines and chemokine analogs, are capable of blocking HIV entry, and studies of their structures and interactions with CCR5 will be key to understanding and optimizing HIV inhibition. The RANTES derivative 5P12-RANTES is a highly potent HIV entry inhibitor that is being developed as a topical HIV prevention agent (microbicide). We have characterized the structure and dynamics of 5P12-RANTES by solution NMR. With the exception of the nine flexible N-terminal residues, 5P12-RANTES has the same structure as wild-type RANTES but unlike the wild-type, does not dimerize via its N-terminus. To prepare the ground for interaction studies with detergent-solubilized CCR5, we have also investigated the interaction of RANTES and 5P12-RANTES with various commonly used detergents. Both RANTES variants are stable in Cymal-5, DHPC, Anzergent-3-12, dodecyltrimethylammonium chloride, and a DDM/CHAPS/CHS mixture. Fos-Cholines, dodecyldimethylglycine, and sodium dodecyl-sulfate denature both RANTES variants at low pH, whereas at neutral pH the stability is considerably higher. The onset of Fos-Choline-12-induced denaturation and the denatured state were characterized by circular dichroism and NMR. The detergent interaction starts below the critical micelle concentration at a well-defined mixed hydrophobic/positive surface region of the chemokine, which overlaps with the dimer interface. An increase of Fos-Choline-12 concentration above the critical micelle concentration causes a transition to a denatured state with a high α-helical content.