The cheA locus of Escherichia coli, which is required for chemotactic behavior, encodes two polypeptide products designated p[cheA]L and p[cheA]S. The mode of synthesis of these two proteins was investigated by transferring various missense and nonsense mutations to a lambda transducing phage and observing the mutant cheA products made after infection of ultraviolet-irradiated host cells. Missense mutations had no effect on either the size or the relative amounts of the two cheA polypeptides. Most nonsense mutations caused premature translational termination of both cheA products, indicating that p[cheA]L and p[cheA]S must be translated from the same coding sequence in the same reading frame. Two exceptional nonsense alleles at the promoter-proximal end of cheA made an intact p[cheA]s but no detectable p[cheA]L. These findings show that the cheA locus may contain two different sites for initiation of translation. The synthesis of both proteins can be effected by the same promoter, but it is not yet clear whether both are translated from identical mRNA molecules. Complementation studies of cheA mutants provided evidence for two functional activities, one associated with the amino terminus of p[cheA]L and the other with the common portions of p[cheA]L and p[cheA]S. It is possible that each cheA product has a different function required for chemotaxis. The possible roles of these two products and the functional significance of bacterial genes with overlapping coding sequences are discussed.