28Mg2+ influx studies in S49 murine lymphoma cells indicate that only 2-3% of total cell Mg2+ content can be exchanged at isotopic equilibrium, implying compartmentation of the newly transported Mg2+. The nature of this compartmentation was examined using selective permeabilization of the plasma membrane with the detergent, digitonin. Control experiments demonstrated that the digitonin permeabilization procedure did not release mitochondrial and lysosomal components, alter mitochondrial respiration, or significantly change cell morphology. Thus, under appropriate conditions, the digitonin permeabilization technique allows determination of the amount of a particular cell constituent within the solute space of the cytoplasm. In nonproliferating cells at an extracellular Mg2+ concentration of 0.1 mM, newly transported Mg2+ equilibrates within 2 h with a small cytoplasmic Mg2+ pool comprising about 3% of the total cytoplasmic Mg2+ (about 2% of total cell Mg2+). The pool of Mg2+ does not equilibrate with bulk cytoplasmic or cellular Mg2+ for at least 16 h. The Mg2+ pool size is dependent on extra-cellular Mg2+ concentration, is saturable with increasing extracellular Mg2+, and reaches a maximal size of 6-7% of total cell Mg2+ at 2 mM extracellular Mg2+. Unlike Mg2+, newly transported Ca2+ is quickly sequestered in noncytoplasmic compartments. In proliferating cells, however, newly transported Mg2+ exchanges extensively with cytoplasmic Mg2+ over the course of 4 h, suggesting that compartmentation of Mg2+ may be dependent on proliferative status.