The levels of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides were found to depend on the concentration of ammonia supplied to photolithoautotrophically grown cultures. Under conditions in which the cells rapidly depleted the available ammonia, the level of in situ RubisCO activity decreased to less than 5% maximum activity; even at its maximum level under these conditions, the RubisCO activity was only 5% of the activity obtained from cultures supplied with saturating levels of ammonia. When cells were incubated with somewhat higher but not saturating amounts of ammonia, in situ RubisCO activity decreased immediately after the cells depleted the cultures of ammonia. The decrease in activity was not due to any detectable degradation of RubisCO protein, indicative of some mechanism to regulate the activity of the enzyme in response to the intracellular levels of assimilated ammonia. Furthermore, under conditions optimum for RubisCO inactivation, in situ RubisCO activity in permeabilized whole cells greatly exceeded the levels of enzymatic activity determined in vitro in cell extracts. Blockage of ammonia assimilation by inhibition of glutamine synthetase with methionine sulfoximine prevented the recovery of form I RubisCO from pyruvate-mediated inactivation, suggesting the presence of regulatory mechanisms common to both CO2 fixation and ammonia assimilation.