Labeling of natural products with biophysical probes has greatly contributed to investigations of their modes of action and has provided tools for visualization of their targets. A general challenge is the availability of a suitable functional group for chemoselective modification. We demonstrate here that an N-terminal ketone is readily introduced into various lanthipeptides by the generation of a cryptic N-terminal dehydro amino acid by the cognate biosynthetic enzymes. Spontaneous hydrolysis of the N-terminal enamines results in alpha-ketoamides that site-specifically react with an aminooxy-derivatized alkyne or fluorophore. The methodology was successfully applied to prochlorosins 1.7 and 2.8, as well as the lantibiotics lacticin 481, haloduracin alpha, and haloduracin beta. The fluorescently modified lantibiotics were added to bacteria, and their cellular localization was visualized by confocal fluorescence microscopy. Lacticin 481 and haloduracin alpha localized predominantly at sites of new and old cell division as well as in punctate patterns along the long axis of rod-shaped bacilli, similar to the localization of lipid II. On the other hand, haloduracin beta was localized nonspecifically in the absence of haloduracin a, but formed specific patterns when coadministered with haloduracin alpha. Using two-color labeling, colocalization of both components of the two-component lantibiotic haloduracin was demonstrated. These data with living cells supports a model in which the a component recognizes lipid II and then recruits the beta-component.