Collagenase dispersed rat liver hepatocytes release Mg2+ when stimulated with norepinephrine or accumulate Mg2+ when stimulated with vasopressin, respectively. Mg2+ fluxes in either direction account for a net loss or gain of approximately 10% of total cell magnesium and are rapidly reversible. Both stimulated Mg2+ efflux and Mg2+ influx require physiological concentration of extracellular NaCl and Ca2+. In the absence of extracellular Na+, Mg2+ efflux, but not influx, can be observed in the presence of extracellular Cl-. Under these conditions, the efflux is inhibited by the Cl-/HCO3- exchanger inhibitor 4,4'-dinitrostilbene-2,2'-disulfonic acid. In hepatocytes, Mg2+ influx, but not efflux, is completely inhibited by thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ ATPase. Several lines of evidence, such as measurements of cytosolic Ca2+ or of cytosolic Ca2+ buffering, indicate that the effect of thapsigargin in inhibiting Mg2+ influx could not be explained by an increase in cytosolic Ca2+. Instead, the inhibition of hepatocyte Mg2+ influx was found to be the result of the depletion of the Ca2+ stored within the endoplasmic reticulum.