We investigated the effects of thyroid hormone treatment on morphology, growth behaviour, and cytoskeletal structures of long-term cultured MC3T3-E1 cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sections. The area covered by the cell and matrix layers (tissue-like area), percent extracellular matrix, average height of tissue-like area, and length and height of single cells were measured histomorphometrically on the cross sections. F-actin was analyzed histochemically and quantitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry. Significant differences between control and T3-treated cells were found after confluency, but not in subconfluent cultures. Control cells continued to proliferate forming multilayers, and produced increasing amounts of extracellular matrix. In contrast, T3-treated cells stopped to proliferate forming two cell layers at the maximum. These cells were flattened, distinctly enlarged, and polygonal in shape. Histochemical staining for F-actin revealed three different staining patterns, depending on the position of the cell within the multilayer of control cultures. Basal cells contained a large number of thick stress fibers in parallel arrangement. Intermediate cells exhibited only a few thick actin filament bundles located at the outermost periphery. The superficial cells were characterized by a large number of thin, parallel-oriented microfilament bundles extending across the entire cytoplasm. The actin pattern of T3-treated cells resembled that of the basal cell layer of the control cells. The amount of F-actin increased with the prolonged T3 treatment. We conclude from these data that the known specific cellular responses to T3 treatment are accompanied by significant morphological alterations indicating pivotal effects of thyroid hormones on osteoblastic differentiation.