An unidentified Streptomyces sp. produces two nucleoside antibiotics, ascamycin and its dealanyl derivative. In contrast to the broad antibacterial activity of dealanylascamycin against various gram-negative and gram-positive bacteria, ascamycin showed selective toxicity against Xanthomonas citri and X. oryzae. Both ascamycin and dealanylascamycin inhibited the protein synthesis of X. citri, but only dealanylascamycin inhibited that of Escherichia coli. In cell-free systems from E. coli and X. citri, both antibiotics, at ca. 0.04 micrograms/ml, inhibited the polyuridylate-directed synthesis of polyphenylalanine by ca. 50%. These data suggest that ascamycin cannot permeate the bacterial membrane. The dealanylating activity toward ascamycin was found only on the cell surface of bacteria susceptible to ascamycin. Dealanylascamycin must then have been transported into cytoplasm, where it inhibited protein synthesis.