The acetate kinase (AckA) and phosphotransacetylase (Pta) pathway provides a central metabolic route for acetate production and utilization in many microorganisms. AckA interconverts acetate and acetyl-phosphate within a pathway that affects diverse cellular processes including growth, motility, chemotaxis and biofilm formation. Here we report unipolar and bipolar localization with distinct fluorescent foci of an AckA-DsRed (red fluorescent protein) fusion protein in Escherichia coli. The fusion protein rescued growth phenotypes caused by an ackA null mutation, demonstrating that polar localized AckA is functional. Polar localization was not altered by cephalexin treatment, suggesting that AckA foci are physically distinct from methyl-accepting chemotaxis proteins. Silencing of the ackA-pta operon using an expressed antisense RNA system did not alter flagella appearance, but inhibited motility and altered biofilm formation. Polar AckA positioning within bacteria may provide gradients of signalling molecules required for directional cellular responses.