Batch-grown Methanobacterium thermoautotrophicum cells grew nonexponentially in the absence of exogenous Pi until intracellular cyclic-2,3-diphosphoglycerate (cyclic DPG) had fallen below 2 mumol/g (dry weight), the limit of detection. Growth resumed immediately upon transfer to medium containing Pi Cyclic DPG levels were also below detection in Pi-limited chemostat cultures operating at a dilution rate of 0.173 h-1 (4-h doubling time), with reservoir Pi concentrations below 200 microM. At this dilution rate, the Pi concentration in the culture was 4 microM. An H2-limited steady state was achieved with 400 microM Pi in the inflowing medium (67 microM in the culture). The cyclic DPG content of these cells was 72 to 74 mumol/g, about one-third the amount in batch-grown cells. The specific growth rate accelerated immediately to 0.36 h-1 (1.9-h doubling time) under washout conditions at high dilution rate. The cellular content of cyclic DPG declined over a 2-h period, and then increased rapidly as the Pi level in the medium approached 200 microM. Expansion of the cyclic DPG pool coincided with a marked increase in Pi assimilation. These results indicated that M. thermoautotrophicum accumulated cyclic DPG only when Pi and H2 were readily available.