The effects of triiodothyronine (T3) on the localization and morphology of alkaline phosphatase (ALP)-positive cells, matrix formation, and apoptosis in MC3T3-E1 cells cultured up to 6 weeks were investigated by light and electron microscopy. Cell size, shape, and frequency of apoptosis were measured histomorphometrically. At all time points both ALP-positive and -negative cells were observed histochemically. Control cultures older than 3 weeks were characterized by colonies of small cuboidal ALP-positive cells. Cross sections revealed that these areas corresponded to unmineralized nodules. The thickening was caused by local accumulation of extracellular matrix. The internodular regions were characterized by ALP-positive spindle-shaped cells randomly distributed throughout all cell layers. Apoptotic nuclei were found within a frequency of 0.2%-1%. With increasing culture time the percentage of apoptotic cells became higher in the nodules. T3 treatment inhibited cell proliferation and stimulated ALP activity. After confluence, T3-treated cultures reached two to three cell layers at maximum and showed a different morphology and histochemical staining pattern. ALP-positive cells were stellar shaped and larger than unstained cells. Small ALP-positive colonies suggested nodule formation; however, the most striking differences between T3-treated and control cultures were a decrease in the amount of extracellular matrix with only few collagen fibers and the absence of local matrix accumulation. Furthermore, the number of apoptotic nuclei was increased. Our data extend beyond previous observations on the role of thyroid hormones in osteoblastic differentiation. Besides their effects on proliferation and cell morphology, they influence ALP activity, matrix composition, nodule formation, and apoptotic transformation.