Thyrotoxicosis is characterised by increased osteoclast activity. Thyroid hormone receptor alpha (TRα) is the predominant TR-isoform in bone and mice lacking TRα have skeletal hypothyroidism with impaired osteoclastic bone resorption. By contrast, mice lacking TRβ have skeletal hyperthyroidism and increased bone resorption. Thus, we hypothesized that T3 acts via TRα to stimulate osteoclastogenesis. Osteoclasts were differentiated in vitro from wild-type (WT), TRα-null and TRβ-null bone marrow precursors using M-CSF and RANKL in the absence or presence of T3 and total RNA was isolated after 1 and 5 days of culture. Numbers of multinucleated tartrate-resistant acid phosphatase (TRAP) positive osteoclasts were quantified and TRAP activity determined at day 5. Data were analysed by ANOVA to determine the effect of genotype on osteoclast parameters. The expression of genes involved in thyroid hormone signalling was investigated by RT-PCR. Precursor cells and mature osteoclasts expressed monocarboxylate transporters 8 and 10 (Mct8, Mct10), the type 2 and type 3 deiodinases (Dio2, Dio3) and TRα1, TRα2 and TRβ1. Despite expression of these key genes, T3-treatment did not affect WT osteoclast numbers (osteoclasts/field 4.3±0.84 vs 3.0±0.78, control versus T3-treatment (n=6)) or differentiation (TRAP activity (mU/ml) 2.8±0.7 vs 2.5±0.7, control versus T3-treatment (n=9)). Similarly, osteoclast cultures from TRα-null and TRβ-null mice were unaffected by T3-treatment. These data demonstrate that T3 does not regulate osteoclastogenesis directly. Nevertheless, increased numbers of mature osteoclasts were generated from TRα-null bone marrow cultures (osteoclasts/field 4.6±0.9 vs 9.3±2.2 vs 2.5±0.7, WT versus TRα-null versus TRβ-null, P<0.05 WT versus TRα-null (n=4)) and TRAP activity was increased 2.5-fold (TRAP activity (mU/ml) 4.4±1.1 vs 11.7±3.6 vs 4.5±2.0, WT versus TRα-null versus TRβ-null, P<0.05 WT versus TRα-null (n=4)). Together with the finding that TRα-null mice have fewer osteoclasts and reduced bone resorption, these data indicate that TRα plays a key permissive role to regulate osteoclastogenesis in vivo.