A single-site mutant of Bacillus subtilis with a rifampin-resistant RNA polymerase has been isolated; this mutation causes temperature-sensitive sporulation. The temperature-sensitive mutation was only expressed during a limited time period, covering the middle third of the sporulation sequence. Mutant cells grown at the nonpermissive temperature exhibited the normal change in RNA polymerase template specificity, accumulated extracellular proteolytic activity and antibiotic activity, but failed to accumulate alkaline phosphatase and, hence, were blocked at or near stage III in the sporulation sequence. Pulse-labeled RNA synthesis was seriously deranged during postexponential growth phase in mutant cells incubated at the nonpermissive temperature.