The surface charge as well as the electrochemical properties and ligand binding abilities of the Gram-positive cell wall is controlled by the d-alanylation of the lipoteichoic acid. The incorporation of d-Ala into lipoteichoic acid requires the d-alanine:d-alanyl carrier protein ligase (DltA) and the carrier protein (DltC). We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DltA with its substrate DltC.