Cell death through apoptosis is a well-known mechanism for maintaining homoeostasis in many developmental and pathological processes. We have recently presented evidence for the occurrence of apoptosis during the formation of bone-like tissue in vitro. MC3T3-E1 osteoblast-like cells in culture develop features of the osteoblastic phenotype and form many cell layers embedded in extracellular matrix which can mineralise. Tri-iodothyronine (T3), even though it enhances the expression of many osteoblastic features, attenuates the multilayer formation to about two layers. The aim of this study was to investigate how T3 prevents multilayer formation. MC3T3-E1 cells were seeded at different densities and cultured for up to 2 weeks. Thereafter we analysed proliferation rate and the distribution of the phases of the cell cycle and studied apoptosis. We found that T3 did not inhibit DNA synthesis. Analysis of the cell cycle phases showed an increase in the number of cells in G0/G1 with increasing cell density, but no significant effect of T3 treatment was found. Morphological investigations showed apoptotic features in both cell layers and culture supernatants. The cells exhibited typical plasma membrane blebbings, chromatin condensation, DNA fragmentation and phagocytosed apoptotic bodies. T3 treatment significantly increased the number of apoptotic cells. We conclude from our data that T3 inhibits multilayer formation of MC3T3-E1 cells by increasing the rate of apoptosis and not by inhibition of proliferation. Because apoptosis is a fundamental regulatory event during bone tissue differentiation, our findings emphasise the importance of thyroid hormones in bone maintenance and development.