Both a correlation and a lack of correlation between guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) level and RNA accumulation have been reported during temperature shifts of E. coli. We have reexamined these phenomena by measuring the total rate of RNA synthesis. After a temperature upshift (23 degrees to 40 degrees C) of E. coli relA+ and relA1 strains, there is an immediate increase in the rate of RNA synthesis which corresponds with the observed in vitro effects of temperature on RNA synthesis (Mangel 1974; Travers 1974). A subsequent increase in ppGpp level is correlated with a decrease in the rate of RNA synthesis. Conversely, following a temperature downshift (40 degrees to 23 degrees C), both relA+ and relA1 bacteria show an immediate decrease in the rate of RNA synthesis. Subsequently all strains studied decrease ppGpp content and correspondingly increase the rate of RNA synthesis after a downshift. By measuring the rate of RNA synthesis we have separated immediate temperature-induced changes in RNA synthesis, from the apparent effects of ppGpp during temperature shifts. As a result, during temperature upshifts and downshifts of relA+, and relA1 bacteria, an inverse correlation between ppGpp content and the total rate of RNA synthesis does exist. The fact that both relA+ and relA1 strains show similar responses to temperature shifts provides additional evidence for the function of relA-independent basal level ppGpp synthesis in regulating RNA synthesis in E. coli.