Two methods were devised to select a series of overlapping deletion mutations carried on episomal elements in Escherichia coli. The deletions were then used in an analysis of (i) the relative position on the genome of previously described mutant loci in the flagellar genes, (ii) the relative position of a newly defined cistron, flaN, and (iii) the orientation and direction of transcription of genes previously assigned to multicistronic transcriptional units. As a result of this analysis and previous work, we report the following arrangement of the flagellar genes in the his-aroD region of the E. coli genetic map: his-flaR-flaQ-flaP-flaA-flaE-flaO-flaC-flaB-flaN-hag-flaD-uvrC-flaI-mot- flaG-flaH. The genes flaA, P, Q, and R, flaB, C, O and E, and flaG and H are co-transcribed in that order.