The turnover number for the sodium-hydrogen exchanger isoform 1 (NHE-1) has been determined in human lymphocytes and MRC5 fibroblasts and in their virally transformed counterparts. Using fluorometric methods, we have determined the intracellular pH and Na+/H+ antiport activity of these cells. Intracellular pH was elevated in both lines of transformed cells. In contrast, Na+/H+ antiport activity was apparently unchanged in simian virus 40-transformed MRC5 fibroblasts (MRC5 SV1 TV1 28.9 +/- 5.2 mM/min compared with MRC5 fibroblasts 26.5 +/- 5.3 mM/min) but slightly increased in Epstein-Barr virus-transformed lymphoblasts (16.7 +/- 1.0 mM/min compared with lymphocytes 13.5 +/- 2.3 mM/min, P < 0.05). With the use of specific antisera to NHE-1, viral transformation was associated with a decreased number of NHE-1 molecules per cell in fibroblasts (from 441,504 +/- 53,428 to 64,745 +/- 7,151 sites/cell, P < 0.001) but an increased number in lymphocytes (from 14,066 +/- 3,100 to 22,474 +/- 4,050 sites/cell, P < 0.01). The NHE-1 density per cell yielded very similar turnover numbers for NHE-1 in the untransformed cells (lymphocytes, 3,161 +/- 833 cycles/s; MRC5 fibroblasts, 3,026 +/- 441 cycles/s), which were significantly elevated in the transformed cells (lymphoblasts, 8,471 +/- 1,177 cycles/s; MRC5 SV1 TV1, 10,521 +/- 2,299 cycles/s, P < 0.001 compared with untransformed cells). We conclude that viral transformation has different effects on Na+/H+ antiport activity and NHE-1 density per cell in different cell types, but the turnover number of NHE-1 is significantly increased after viral transformation, which correlates with the increased proliferation rate of these transformed cells.