Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.