Thyroid hormone regulates the expression of G protein in tissues such as fat and heart. In the brain, very little information is available relative to the regulation by thyroid hormone of G proteins. Here, we show that the expression of the Galphai1 gene is induced by thyroid hormones in the rat cerebellum during development. Hence, the levels of Galphai1 transcripts and protein were decreased in the cerebellum of hypothyroid neonates. In situ hybridization studies showed that the neurons of the cerebellar cortex, particularly Purkinje cells, were affected. Surprisingly, and in contrast with the in vivo stimulatory effect described above, thyroid hormone repressed the activity of the rat Galphai1 promoter in vitro, suggesting that the effect of this hormone in the cerebellum is indirect. In this regard, we present data suggesting that the transcription factor C/EBPbeta could be implicated. First, there are active CEBP binding sites in the Galphai1 promoter. Second, we have found a diminished DNA binding activity of hypothyroid nuclear proteins to a Galphai1 promoter sequence containing a C/EBP binding site. Third, this complex is likely to contain C/EBPbeta protein as it is displaced by specific anti-C/EBPbeta antibodies. Finally, there is a significant decrease in the C/EBPbeta protein content in the hypothyroid cerebellar cortex.