The cloning and molecular genetic analysis of a locus mapping within the flagellar gene (fli) complex of Salmonella typhimurium is reported. A copy of the insertion element IS200 was located in a noncoding stretch of DNA upstream of the fliA gene. Comparative nucleotide sequence analysis showed that this copy of IS200 was 711 bp long and that its flanking regions contained no features common to other characterized insertion sites of this element. The element was located 37 bp downstream of an ORF whose product was shown by interspecific transfer and amino acid analysis to carry out N-methylation of selected lysine residues in Salmonella flagellin. The sequence and phenotype of this ORF identified it as fliB, encoding the only prokaryotic N-methylase acting on amino groups to have been characterized to date. It was found to be conserved among all clinically significant serovars of Salmonella. The IS200 insertion site is of particular interest since it was conserved in all but two rare evolutionary lines of S. typhimurium, and was absent from 85 Salmonella strains belonging to 37 other serovars. It is thus a phylogenetically significant marker at the serovar level.