During the inhibition of RC(str), but not RC(rel) mutants of Escherichia coli by trimethoprim the unusual nucleotides MSI (guanosine tetraphosphate, ppGpp) and MSII rapidly accumulated. The production of these nucleotides was not dependent on the addition of nucleotide base supplements to RC(str) cultures before trimethoprim, and the MSI nucleotide concentrations in non-supplemented or purine-supplemented cultures were comparable with the concentrations obtained when the cells were inhibited with l-valine (1g/l). Rifampicin rapidly decreased MSI and MSII nucleotide concentrations in trimethoprim-inhibited cultures to the basal values. Several situations were noted, in which MS nucleotide concentrations in trimethoprim-inhibited RC(str) cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RC(str) mutants were first inhibited with trimethoprim and then given purines 15min later, MS nucleotide concentrations fell rapidly, because of a temporarily enhanced rate of accumulation of stable RNA. However, after a further 5min, RNA accumulation stopped, though MS nucleotide concentrations remained low. Also, if either glycine or methionine were added to trimethoprim-inhibited cultures supplemented with purines, RNA accumulation did not resume, though MS nucleotide concentrations rapidly declined. With both amino acids present, there was both a decline in MS nucleotide concentration and a resumption in stable RNA synthesis. These findings suggest that MSI nucleotide concentrations in trimethoprim-inhibited bacteria are not the sole factors in the control of stable RNA synthesis. It is possible that, during the period when the RC(str) cells contained high concentrations of MS nucleotides, some factor important in the MSI-mediated control of stable RNA synthesis was irreversibly inactivated. However, as antibiotics (e.g. chloramphenicol) both abolished high MS nucleotide concentrations and permitted a rapid resumption of stable RNA accumulation in the same conditions, it is more likely that an additional control mechanism has come into play.