Assimilation of nitrogen requires the synthesis of only two central intermediates, glutamate and glutamine, from which other compounds derive nitrogen by secondary transfers. We measured the internal pool sizes of glutamate and glutamine in Salmonella typhimurium under conditions of external nitrogen limitation or sufficiency. When growth was slowed by nitrogen limitation, the glutamine pool was lower by a factor of up to 10, whereas the glutamate pool remained high. The decrease in the glutamine pool was general in nature, being seen with various limiting nitrogen sources in batch culture and with ammonia, the optimal nitrogen source, as the limiting nutrient in continuous culture. The only nitrogen source that gave discordant results was alanine, and we present evidence that alanine has inhibitory effects which extend beyond simple nitrogen limitation. Studies with mutant strains having altered nitrogen assimilation indicated that the decreases in the glutamine pool observed in the wild-type strain under nitrogen-limiting conditions were probably sufficient to account for slow growth and were likely to be responsible for slow growth. Hence we postulate that external nitrogen limitation is first perceived by Salmonella as a drop in its internal glutamine pool.