To study the relation between activation of the Na(+)-H+ antiporter and gastrointestinal cell proliferation, we characterized this antiporter in a pancreatic cell line (AR42J) and studied the effects of mitogenic and nonmitogenic agents as well as those of Na(+)-H+ exchange blocking agents on DNA synthesis. Characteristics of amiloride-sensitive Na+ uptake were those of the Na(+)-H+ exchanger: 1) Na+ uptake was increased by intracellular acidification and depended on external [Na+] and pH; 2) concentrations for half-maximal inhibition (IC50) of Na+ uptake (3 mM [Na+] in medium) were 40 nM 5-(N,N-hexamethylene)amiloride (HA) less than 0.1 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA) less than 1 microM 5-(N,N-dimethyl)-amiloride (DMA) less than 40 microM amiloride; 3) IC50 for amiloride and analogues to inhibit Na+ uptake depended on [Na+] in medium (in 25 mM Na+ medium, the IC50 values were higher than in 3 mM and were 1 microM EIPA less than 10 microM DMA less than 0.3 mM amiloride). Growth factors for AR42J cells (dialyzed fetal calf serum and epidermal growth factor) activated Na+ uptake in a dose-dependent manner. Bombesin, which is nonmitogenic for AR42J cells, also increased Na+ uptake, indicating that activation of the antiporter is not sufficient to initiate cell proliferation in the AR42J cell line. The effects of Na(+)-H+ exchange blocking agents were tested on serum-stimulated cell proliferation. Decreasing external Na+ dramatically decreased AR42J cell proliferation. Amiloride and analogues inhibited [3H]thymidine incorporation in the same range of concentrations as that with which they inhibited Na(+)-H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)