AbstractThe conformations of two synthetic pentapeptides with antimicrobial activity and their 4‐fluorophenylalanine (Pff)‐containing analogues (ArXArXAr‐NH2; Ar=Phe, Pff; X=Lys, Arg) have been studied. NMR experiments carried out both in aqueous fluoroalcohol solutions and SDS micelles permitted their interactions with membrane‐like environments to be explored. WaterLOGSY experiments and Mn2+‐based paramagnetic probes were also applied to assess their orientations with respect to the SDS micelles. In addition, pulse‐field gradient (PFG) diffusion NMR spectroscopy studies were conducted, under different experimental conditions (i.e., concentration, temperature) to characterize the possible changes in the peptides' aggregation states as a putative critical factor for their antimicrobial activity. Finally, molecular dynamics simulations on a variety of conformations showed the intrinsic flexibility of these peptides in both aqueous solutions and membrane‐mimetic systems.