Primary rat osteoblast-like cells (Ob) were grown under strictly serum-free conditions for up to 20 days. In the presence of triiodothyronine (T3) at concentrations of 0.01 and 0.1 nM, Ob proliferation was enhanced. Moreover, a decrease of alkaline phosphatase (AP) activity, a differentiation marker for Ob, was prevented, whereas protein synthesis (collagen and noncollagen protein) was decreased. T3 at much higher concentration (10 nM) had no significant effect on cell proliferation and matrix formation but decreased AP activity disproportionately. Thus, T3 at close to physiological concentrations stimulates growth and maintains differentiation of Ob.