The movement of nucleophosmin from nucleoli to nucleoplasm in HeLa cells induced by cytotoxic drugs and detected by immunofluorescence is inhibited by concomitant treatment with antimycin A in glucose-free medium. Incubation of HeLa cells with antimycin A (300 nM; 30 min) and glucose-free medium resulted in an approximately 90% decrease in cellular ATP pools. To study the biochemical events involved in nucleophosmin translocation, we used an in vitro system consisting of Triton-permeabilized HeLA cells. Incubation of permeabilized cells with ATP (0.5 mM; 1 h) resulted in the translocation of nucleophosmin from nucleoli to nucleoplasm and cytoplasm. Similarly to drug-induced nucleophosmin translocation in whole cultured cells, there is no reduction (measured by e.l.i.s.a.) or degradation of nucleophosmin or change in the ratio of the high-molecular-mass form to the monomeric form (ascertained by Western blotting) during ATP treatment of permeabilized cells. Together, these results indicate a requirement for ATP for redistribution of nucleophosmin from nucleoli to nucleoplasm. Because this permeabilized cell model is simple and efficient and works effectively with exogenous factors, it should provide a powerful tool for investigating the biochemical features of nucleophosmin translocation from nucleoli to nucleoplasm.