The effect of 5'-O-diphosphorylphosphonylmethylribonucleosides I on the transcription reaction catalyzed by DNA-dependent RNA polymerase from Escherichia coli was studied. These analogues of ribonucleoside 5'-triphosphates are not enzyme substrates but inhibit the transcription reaction, competing specifically with the natural substrates. According to the inhibition constants, the pyrimidine derivatives Ia,b are more potent inhibitors than the purine derivatives Ic,d.