NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na(+)/H(+) exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (V(max)) by (22)Na(+) uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 +/- 3.5, 64.6 +/- 3.3, 20.0 +/- 2.6, and 14.0 +/- 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 +/- 9.6, 92.1 +/- 8.6, and 99.2 +/- 9.1 s(-1), when V(max) was determined using (22)Na uptake at 22 degrees C and 742 +/- 47, 459 +/- 16, and 609 +/- 39 s(-1) when V(max) was determined using fluorometry at 37 degrees C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.