Synthesis of single-stranded DNA of bacteriophage M 13 is blocked by rifampicin. This inhibition is not observed in an Escherichia coli mutant possessing an RNA polymerase resistant to rifampicin. Since rifampicin stops single-strand synthesis faster than chloramphenicol, inhibition by rifampicin does not seem to result from a transcriptional block of protein synthesis, particularly in view of the long half-life of the messenger RNA for M 13. It is, therefore, concluded that the interaction of rifampicin with RNA polymerase directly affects single-strand DNA synthesis. Possible mechanisms are discussed.