Chlorosis-inducing isolates of Xanthomonas albilineans, the sugarcane leaf scald pathogen, produced a mixture of antibacterial compounds in culture. The antibiotic mixture, which eluted as a single strongly retarded peak from Sephadex LH-20 in methanol, was bactericidal to Escherichia coli. Inhibition of E. coli was not reversed by added nutrients, and affected cells were not lysed but many accumulated polyphosphate granules. The major antibacterial component, isolated in crystalline form after HPLC, is given the trivial name albicidin. Near the minimum inhibitory concentration, albicidin caused a complete block to DNA synthesis, followed by partial inhibition of RNA and protein synthesis, as assessed by incorporation of radioactive precursors. Spontaneous antibiotic-resistant mutants of E. coli showed no cross-resistance between albicidin and inhibitors of either subunit of DNA gyrase. Mixing albicidin with purified DNA from E. coli did not alter the thermal denaturation behaviour of the DNA, or the absorption spectrum of the antibiotic. PolA+ and PolA - strains of E. coli were equally sensitive to albicidin, indicating that the antibiotic does not bind to or modify DNA. Selective inhibition of DNA synthesis without evidence of DNA binding suggests a specific interaction of albicidin with an essential replication protein.