Escherichia coli bearing an rpoS amber or disrupted mutation exhibited a significant decrease in the number of colony-forming units (c.f.u.) when exposed to nitrogen starvation, which was not observed in cells bearing a functional rpoS allele. The decrease in the number of c.f.u. that was observed about 25 h after initiation of nitrogen starvation was prevented by the addition of nitrogen within 3 h but not by the addition of nitrogen at more than 7 h after the initiation of nitrogen starvation, suggesting that a process leading to a decline in c.f.u. starts within this period. DNA microarray analysis of the rpoS mutant showed that a large number of genes including many functionally undefined genes were affected by nitrogen starvation. The expression levels of sigma(S) and sigma(H) regulon genes encoding acid-resistant proteins (hdeA, hdeB, gadA and gadB), DNA-binding protein (dps), chaperones (dnaK, ibpA, ibpB, dnaJ and htpG), chaperonins (mopB and mopA) and energy-metabolism-related proteins (hyaABCDF and gapA), and those of other genes encoding nucleotide-metabolism-related proteins (deoC and deoB), cell-division protein (ftsL), outer-membrane lipoprotein (slp) and DNA-binding protein (stpA) were significantly decreased by 10 h nitrogen starvation. The genes encoding transport/binding proteins (nac, amtB, argT, artJ, potF and hisJ) and amino acid-metabolism-related proteins (glnA, trpB, argG, asnB, argC, gdhA, cstC, ntrB, asd and lysC) were significantly up-regulated under the same condition, some of which are known Ntr genes expressed under nitrogen limitation. On the basis of these results, possible causes of the decrease in the number of c.f.u. under nitrogen starvation are discussed.