Daptomycin at the MIC allowed the cell mass increase of enterococcal strains and Bacillus subtilis to continue for 2 to 3 h at rates comparable to those of the controls. During this time the cell shape of the former changed to a rod configuration and that of the latter changed to long rods. In these bacteria, in which cell mass continued to increase, the MIC of daptomycin inhibited peptidoglycan synthesis by no more than 20% after 20 min of incubation and by roughly 50% after 2 h of incubation. Other macromolecules, such as DNA, RNA, and proteins, were only slightly affected. In contrast, incorporation of [14C]acetate into lipids was reduced by about 50% in the various strains after 20 min of treatment with daptomycin at the MIC. When the effect of the major lipid-containing polymers on synthesis was evaluated in detail, it was found that under conditions in which peptidoglycan and the other macromolecules mentioned above were inhibited only slightly (20%) and total lipid synthesis was inhibited by 50%, synthesis of teichoic and lipoteichoic acid was inhibited by 50 and 93%, respectively. Daptomycin was not found to enter the cytoplasm of either bacterial or mammalian cells. It bound, in the presence of calcium ions only, to whole bacterial cells, cell walls (both those that contained and those that did not contain membranes), and isolated membranes of bacterial and mammalian cells. Washing with EDTA removed daptomycin from all cells mentioned above and cell fractions except the bacterial membrane. It is concluded that lipoteichoic acid is most likely the primary target of daptomycin.