This study was initiated to investigate the mechanism of ceruloplasmin (CP)-mediated iron release from brain cells using BT325 cells (a glioblastoma cell line); however, negative results were obtained. The BT325 cells were preloaded with 1 microM 59Fe2+ in sucrose (pH 5.8) for 60 min, and then with CP (0-300 microg/ml) for 30 min at 37 degrees C. The addition of CP, either at low (25 microg/ml) or high (300 microg/ml) concentrations, did not lead to a significant change in iron release from iron-loaded BT325 cells. No significant difference in total iron of cells was found between all CP treated groups and the control (P>0.05). Although apotransferrin (apoTf) was shown to have a role in iron release from the cells, the effect of apoTf was not significantly affected by the addition of different concentrations of CP. When the cells were incubated with 1 microM 59Fe2+ in the presence of varying amounts of CP for 30 min at 37 degrees C, it was found that CP increased iron uptake. The total iron uptake by BT325 cells in CP treatment groups (25, 75, 150, 300 microg/ml) was significantly higher than that in the control (no CP addition) (all P<0.01). Furthermore, in contrast to our expectation, CP was shown to promote significantly iron uptake in not only iron-sufficient but also iron-deficient cells. These results showed that CP had a role in iron uptake rather than release in BT325 cells.