By introduction of deletions and/or insertions, we have defined a small DNA fragment (1.58 kilobase pairs) which contains the ant gene. Thus, transformants of all plasmid constructs containing this segment exhibit Antup phenotype, i.e. growth is Li+-resistant and Na+/H+ antiporter activity is increased in isolated everted membrane vesicles. Utilizing the T7 promoter expression system, we also found that this fragment encodes for a single protein of 35 kDa. The DNA fragment has been sequenced and found to contain an open reading frame of 1085 base pairs. Analysis of the sequence of the predicted protein suggests the presence of 10 putative transmembrane segments in the protein.