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This report describes the first synthesis and application of a fluorescent teixobactin analogue that exhibits antibiotic activity and binds to the cell walls of Gram-positive bacteria. The teixobactin analogue, Lys(Rhod)(9),Arg(10)-teixobactin, has a fluorescent tag at position 9 and an arginine in place of the natural allo-enduracididine residue at position 10. The fluorescent teixobactin analogue retains partial antibiotic activity, with minimum inhibitory concentrations of 4–8 μg/mL across a panel of Gram-positive bacteria, as compared to 1–4 μg/mL for the unlabeled Arg(10)-teixobactin analogue. Lys(Rhod)(9),Arg(10)-teixobactin is prepared by a regioselective labeling strategy that labels Lys(9) with an amine-reactive rhodamine fluorophore during solid-phase peptide synthesis, with the resulting conjugate tolerating subsequent solid-phase peptide synthesis reactions. Treatment of Gram-positive bacteria with Lys(Rhod)(9),Arg(10)-teixobactin results in septal and lateral staining, which is consistent with an antibiotic targeting cell wall precursors. Concurrent treatment of Lys(Rhod)(9),Arg(10)-teixobactin and BODIPY FL vancomycin results in septal co-localization, providing further evidence that Lys(Rhod)(9),Arg(10)-teixobactin binds to cell wall precursors. Controls with either Gram-negative bacteria, or an inactive fluorescent homologue with Gram-positive bacteria, showed little or no staining in fluorescence micrographic studies. Lys(Rhod)(9),Arg(10)-teixobactin can thus serve as a functional probe to study Gram-positive bacteria and their interactions with teixobactin.