To identify thyroid hormone-sensitive neuronal populations in the forebrain, we studied the effects of thyroid hormone deficiency and replacement on the expression of RC3 messenger RNA (mRNA) in the rat brain by in situ hybridization. RC3/neurogranin is a brain-specific, calmodulin-binding, protein kinase C substrate that has been implicated in postsynaptic events involving calcium as a second messenger. We have previously shown that RC3 mRNA and protein concentrations are thyroid hormone dependent in developing and adult rats. In normal developing rats, RC3 expression occurs in two phases. Before postnatal day 10 (P10), RC3 mRNA was detected mainly in layers II/III and V of cerebral cortex and the CA fields of the hippocampus. From P10 to P15, it decreased in layer V and increased in layer VI, the retrosplenial cortex, the caudate-putamen nucleus, and the dentate gyrus. Expression in the caudate followed a lateral to medial gradient. Thyroid hormone deficiency interfered with the late phase of RC3 expression, such that developing hypothyroid rats showed lower RC3 expression in layer VI, the retrosplenial cortex, the dentate gyrus, and the caudate, and increased expression in layer V. These changes were reverted by T4 treatment. Adult- onset hyperthyroidism also reversibly decreased hybridization in the striatum. In contrast to other molecular targets of thyroid hormone in the brain, such as myelin genes, expression of RC3 was also affected by long term hypothyroidism in the absence of hormone replacement, indicating that thyroid hormone is a required factor for the cell-specific control of RC3 expression. In addition to identifying thyroid hormone-sensitive neurons, our results suggest that one action of thyroid hormone during brain development is the timely coordination of gene expression among phenotypically different, region-specific neuronal populations.