These studies examined the effects of protein kinase C activation and calmodulin inhibition on the amiloride-sensitive NHE-1 isoform of the Na+- H+ exchanger in defined host cells. Our objective was to define differences in the cellular regulatory responses using a specified isoform of the Na+- H+ exchanger. Suspended cells were loaded with 2',7'-bis(carboxyethyl)-5,6- carboxyfluorescein (BCECF) and preacidified to a cytosolic pH of 6.2. Wild- type mouse Ltk- cells, human A-431 cells, and mutant mouse fibroblasts stably transfected with the human NHE-1 isoform (LAP+ cells) were examined to define the maximal rate of transport (V(max)) in response to 140 mM external Na+, the Hill stoichiometric coefficient, and the cytosolic pH at which the NHE-1 isoform was half-maximally stimulated (pH50). The mouse NHE-1 isoform had a greater affinity for cytosolic H+ than the human NHE-1 isoforms. Calmodulin antagonism with N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide reduced the V(max) and shifted the pH50 in the acidic direction, especially in the A-431 cells. Protein kinase C stimulation had a similar effect in A-431 cells and little effect in the wild-type (Ltk-) and transfected (LAP+) mouse cells. While the NHE-1 isoform contains several potential phosphorylation sites, the cellular milieu in which the isoform is expressed has an important effect on the modulation of NHE-1 activity.