The Mg content of axons freshly dissected from living specimens of the tropical squid Doryteuthis plei was determined by atomic absorption spectroscopy to be 4.2 +/- 0.2 mmol/kg axoplasm. The axon's ability to maintain this physiological content of total intracellular Mg([Mg]i) was studied. Mgi was shown to be a linear function of Mgo when Mgo of incubating fluid was varied between 0 and 250 mM. When Mgo = 15 mM, Mgi was found to be the same in incubated fibers as in fibers freshly dissected. Mgi levels were unaffected by depolarization of the membrane by high Ko. Stimulation resulted in an extra influx of Mg of 0.05 pmol/(cm2 . impulse) when Mgo = 55 mM. Mgi was found to be a complicated function of the concentration of extracellular Na or Li (Xo), which was substituted for Tris. With 385 mM Lio the Mgi level was found to be 2.5-fold larger than the level observed with 385 mM Nao after incubation for 3 h. The function relating Mgo to Xo was qualitatively unaffected in axons poisoned with the mitochondrial uncoupler carbonyl cyanide, p-trifluorome-thoxy-phenylhydrazone (FCCP) and the inhibitor of glycolysis, iodoacetic acid (IAA); the absolute levels of Mgi, however, were some 30% higher in the poisoned axons at all [X]o explored. 2 h incubation of axons in a 333 mM Mg, 40 mM Li solution increased Mgi 3.5-fold in control axons and 5-fold in poisoned axons. These Mg-loaded axons were able to recover physiological levels of Mgi with a half-time of 3-5 h only if kept in a solution which contained Na (220 mM) regardless of whether the axons had been inhibited with FCCP + IAA. Therefore, it may be concluded that the physiological Mgi concentration can be maintained by the Na electrochemical gradient, even when the axon is metabolically poisoned.