The authors have identified a fourth flagellin gene in a 21850 bp region of the Agrobacterium tumefaciens C58C1 chromosome containing at least 20 genes concerned with flagellar structure, assembly and function. Three flagellin genes, flaA, flaB and flaC, orientated rightward, are positioned in a tandem array at the right end, with the fourth, substantially larger gene, flaD, in the opposite orientation, at the left end. Between these lie four apparent operons, two transcribed in each direction (motA, flhB leftward; flgF, flgB rightward) from a divergent position approx 7.5 kb from the left end. This unifies the previously published motA, flgB and flaABC sequences into a single region, also containing the homologues of flhB, flgF and fliI. Site-specific mutagenesis of fliI results in a non-flagellate phenotype, while a Tn5-induced flhB mutant possesses abnormal flagella. Mutagenesis and protein profiling demonstrate that all four flagellins contribute to flagellar structure: FlaA is the major protein, FlaB and FlaC are present in lesser amounts, and FlaD is a minor component. FlaA has anomolous electrophoretic mobility, possibly due to glycosylation.