A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated. The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+. The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal. Judging from the reversion frequency, the defect seems to be due to a single mutation. The mutant could not grow at alkaline pH. Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium. This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.