Iron overload occurs frequently in thalassemia as a consequence of regular blood transfusions, and iron has been found to accumulate in bone, but skeletal toxicity of iron is not clearly established. In this study, bone biopsies of thalassemic patients were investigated by light (n = 6) and electron microscopy (n = 8) in order to analyze iron distribution and possible iron-associated cellular lesions. Sections (5 microns thick) were used for histomorphometry and iron histochemistry. Ultrathin sections were examined with an energy filtering transmission electron microscope. Iron was identified by electron energy loss spectroscopy (EELS), and iron distribution was visualized by electron spectroscopic imaging (ESI) associated with computer-assisted treatment (two-window method). This study shows that EELS allows the detection of 4500-9000 iron atoms, and that computer-assisted image processing is essential to eliminate background and to obtain the net distribution of an element by ESI. This study shows also that stainable iron was present along trabecular surfaces, mineralizing surfaces, and on cement lines in the biopsies of all patients. Moreover, iron was detected by EELS in small granules (diffusely distributed or condensed in large clusters), in osteoid tissue, and in the cytoplasm of bone cells, but not in the mineralized matrix. The shape and size (9-13 nm) of these granules were similar to those reported for ferritin. As for iron toxicity, all patients had osteoid volume and thickness and osteoblast surface in the normal range. Stainable iron surfaces did not correlate with osteoblast surfaces, plasma ferritin concentrations, or the duration of transfusion therapy.(ABSTRACT TRUNCATED AT 250 WORDS)