Despite the recognized importance of thyroid hormones for normal brain development, little is known about the critical molecular events underlying this role. We investigated the molecular basis of thyroid hormone action on the developing brain by comparing genome-wide gene expression patterns in the cerebellum between euthyroid and hypothyroid juvenile mice using microarrays. Pregnant dams were treated with 0.1% or 0.04% 6-propyl-2-thiouracil (PTU) in drinking water continuously from day 13 post conception until weaning to produce hypothyroid pups. Cerebella were collected from vehicle and 0.1% PTU treated pups at post-natal day (PND) 15, and mRNA from these was subjected to microarray analysis using Agilent high-density oligonucleotide chips. Statistical analysis (MAANOVA) revealed significant differential expression in 2940 genes including 1357 up- and 1583 down-regulated genes. Further analysis (combined MAANOVA and ANOVA) identified 204 significantly altered genes. Hypothyroidism had a greater effect on gene expression in male than in female pups. Transcriptional changes in several genes [Syt12 (Synaptotagmin 12), Rcor (REI-silencing transcription factor co-repressor), Bag3 (Bcl-associated athanogene 3), p21, cyclin D, Bax (Bcl2-associated X protein), and Pcp2 (Purkinje cell protein 2)] were confirmed using real-time (RT) PCR analysis. Significantly altered expression of Bag3 in cerebella from PND 15 and PND 60 pups exposed to PTU suggests permanent functional alterations in the hypothyroid brain. The thyroid hormone negative regulation of Rcor expression was confirmed in vitro using HepG2 cells. In addition to Rcor, expression of several other genes that code for critical components of the REST (REI-silencing transcription factor) pathway was shown to be altered in hypothyroid animals. These results suggest that modification of this pathway may have a significant role in causing impaired development in the hypothyroid brain. Crown copyright (c) 2005. Published by Elsevier Inc. All rights reserved.