Thyroid hormones stimulate bone turnover in vivo and increase Ca release from bone in vitro. To investigate further the effects of thyroid hormones in bone, we have characterized specific nuclear receptors for triiodothyronine (T3) in neonatal mouse calvaria. Maximal specific binding of [125I]T3 to isolated nuclei occurred within 60 min at 22°C. [125I]T3 binding was completely and rapidly displaced by the addition of 10−6 M unlabeled T3; the dissociation appears to be first order with t1/2 = 36 min. The IC50 for competition by unlabeled T3 was approximately 10−8 M. The relative affinity of thyroxine (T4) for the receptor was ˜10 × lower than T3, consistent with its lower biological potency in most target tissues for thyroid hormones. Only weak competition was observed with diiodotyrosine at concentrations up to 10−4 M. We have previously shown that the cardiotonic agent milrinone stimulates bone resorption in vitro with characteristics similar to those of T4. Structural homology between milrinone and T4 was recently reported. Milrinone, like diiodotyrosine, was only a weak competitor for binding at concentrations up to 10−4 M. Milrinone inhibited collagen synthesis in the calvaria. The results suggest that the effects of milrinone on bone turnover in calvaria in vitro are probably not mediated through a thyroid hormone receptor.