A conditional-lethal mutant was isolated as having a flagellar regulatory phenotype at 30 degrees C and being unable to grow at 42 degrees C. Chromosomal mapping localized the mutation to the serT gene, which encodes an essential serine tRNA species (tRNA((cmo)5UGA)(Ser)). DNA sequence analysis revealed the mutation to be a single base change in G:A at position 10 of the serT gene that lies within the D-stem of the essential tRNA((cmo)5)UGA(Ser) species. tRNA((cmo)5)UGA(Ser) recognizes UCA, UCG, and UCU codons, but UCU is also recognized by tRNA(GGA)(Ser) and UCG by tRNA(CGA)(Ser). No other tRNAs are known to read the UCA codon. Thus, the UCA codon is specifically recognized by tRNA((cmo)5)UGA(Ser). We show that the anti-sigma(28) activity of FlgM is defective in the serT mutant strain. The serT allele causes a 10-fold increase in sigma(28)-dependent fliC promoter transcription, indicating a defect in FlgM anti-sigma(28) activity in the presence of the serT mutation. The flgM gene contains only one UCA codon. Changing the UCA of flgM to ACG reversed the effect of the serT allele. Implications for context effects in regulation of gene expression are discussed.