The element compartmentation in cultured rat hepatocytes was studied by electron probe X-ray microanalysis of freeze-dried cryosections after exposure of the cells to the ionophores valinomycin, ionomycin or gramicidin A. The most striking effect of these ionophores is the decrease of the intracellular potassium/sodium ratio from values of approximately 10 under control conditions to values below I after application of the ionophores. Changes of sodium, potassium and chloride are similar in cytoplasm and nucleus. However, elemental changes are delayed or impeded in mitochondria with respect to the surrounding cytoplasm. The water portion of cytoplasm and mitochondria slightly increases. Besides that, each ionophore has specific effects on the intracellular ion distribution. As compared to gramicidin A and ionomycin, valinomycin does not change the intracellular chloride content. lonomycin induces calcium accumulation in mitochondria. The cytotoxic effects of the studied ionophores on the intracellular element distribution are more complex than supposed from their ion selective properties in membranes. (C) 2002 Elsevier Science Ltd. All rights reserved.