Effects of extracellular magnesium ions ([Mg2+]o) on intracellular free Mg2+ ([Mg2+]i) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg2+ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg2+]o, [Mg2+]i in yeast cells was 0.91 +/- 0.08 mM. Elevation of [Mg2+]o to 1.97 mM induced rapid (within 5 min) increments in [Mg2+]i (2.18 +/- 0.11 mM). Lowering [Mg2+]o to 0.06 mM, however, exerted no significant effects on [Mg2+]i (0.93 +/- 0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg2+]o used, the subcellular distribution of [Mg2+]i remained heterogeneous, i.e. where the sub-plasma membrane region > cytoplasm > nucleus. [Mg2+] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg2+] when placed in 1.97 mM [Mg2+]o. We conclude that [Mg2+]i in fission yeast is maintained at a physiologic level when [Mg2+]o is low, but intracellular free Mg2+ rapidly rises when [Mg2+]o is elevated. Like most eukaryotic cells, yeast may have a Mg2+ transport system(s) which functions to maintain gradients of Mg2+ from the outside to inside the cell and among its subcellular compartments.