In mouse thymocytes, a stereospecific saturable energy-dependent and ouabain-inhibitable system facilitates T3, but not T4, entry. We studied here the effect of sodium depletion on cellular uptake of thyroid hormones by mouse thymocytes. Time-course experiments indicated that extracellular sodium depletion reduced [125I]T3 uptake at each time studied. At equilibrium, the removal of extracellular sodium and its substitution with isoosmotic choline decreased saturable [125I]T3 uptake by 60 +/- 10%; this effect was dose dependent. The substitution of sodium with lithium, instead of choline, had no effect on the uptake process. [125I]T4 uptake was lower than that of [125I]T3 and not affected by sodium depletion. The half-maximal effect of sodium deprivation on [125I]T3 uptake was reached at an extracellular sodium concentration of about 40 mM. The variation of external pH influenced T3 accumulation by thymocytes. [125I]T3 progressively decreased from acid to alkaline pH under normal and sodium-depleted conditions; however, the sodium-dependent fraction was more than doubled at physiological pH compared to that at more acidic and more alkaline pH. The sodium ionophore monensin decreased T3 uptake by 51% at a concentration of 20 microM. These results indicated the existence of a sodium-related mechanism of T3 uptake into mouse thymocytes that does not operate for T4 uptake.