Loperamide, an opiate agonist, inhibited the Na+-H+ exchanger in brush-border membrane vesicles isolated from term human placenta, rabbit renal cortex and rabbit small intestine in a dose dependent manner. Because the placental Na+-H+ exchanger was the most sensitive to inhibition by loperamide (IC50 = 60 μM), characterization of the inhibition was done with the placental Na+-H+ exchanger. The inhibition of the placental Na+-H+ exchanger by loperamide was instantaneous and freely reversible. Kinetic analyses demonstrated that the inhibition was of a mixed type. Loperamide (70 μM) reduced the maximal velocity (V(max)) from 46.4 ± 1.2 to 34.5 ± 1.1 nmol/mg of protein/15 s and increased the apparent affinity constant (K(t)) for Na+ from 12.3 ± 1.0 to 16.5 ± 1.5 mM. Loperamide interacted with the exchanger protein at more than one site. The effects of loperamide were not antagonized by naloxone, suggesting the noninvolvement of opiate receptors in this process. These results differentiate loperamide from other Na+-H+ exchanger inhibitors such as amiloride, cimetidine and clonidine, which interact with the exchanger at a single site in a strictly competitive manner.