The Na+/H+ exchanger isoform NHE1 is localized to the basolateral membrane of renal and intestinal epithelia. We examined the plasma membrane distribution, biosynthesis, and cell surface delivery of NHE1 in A6 epithelia. NHE1 was localized to the basolateral membrane. Studies of NHE1 biosynthesis with a pulse-chase protocol demonstrated that a core glycosylated, endoglycosidase H-sensitive, 90-kDa NHE 1 was present 0-5 h into the chase period and that mature 110-kDa NHE1 was present 1-24 h into the chase period. Studies of plasma membrane delivery of newly synthesized NHE1 demonstrated that the 90-kDa NHE1 was detected at both apical and basolateral membranes 2-5 h into the chase period. The 110-kDa NHE1 was observed at the basolateral membrane 5-24 h into the chase period. These results suggest that NHE1 is expressed primarily at the basolateral membrane of A6 cells, that core glycosylated NHE1 is delivered to the plasma membrane in a nonpolarized manner, and that mature 110-kDa NHE1 is delivered to the basolateral membrane.