Comparison of respiration-driven active transport of alkali cations from E. coli cells loaded with Na+ or Li+ showed that Li+ could not be expelled from the cells like Na+. K+ accumulation, which was fast in Na+-loaded cells, was strongly inhibited in Li+-loaded cells, despite high membrane potential and respiratory rate. When Li+-loaded cells were placed into medium containing Na+ instead of Li+, Li+/Na+ exchange took place initially, while K+ accumulation was delayed. Only after almost all inside Li+ was replaced by Na+ did active Na+ and K+ transport commence. These data confirm that it is a distinct active sodium transport system (AST) with Na+,K+/H+ antiporter activity, and not the Na+/H+ antiporters, that is responsible for active Na+ transport in E. coli [Verkhovskaya, M.L., Verkhovsky, M.I. and Wikstrom, M. (1996) Biochim. Biophys. Acta 1273, 207-216]. In contrast to the Na+/H+ antiporters, the AST system is inhibited by Li+.