Thyroid hormone is required for basal and estrogen-induced expression of anterior pituitary galanin. Steady-state anterior pituitary galanin mRNA levels decreased 6-fold in hypothyroid rats after 3 weeks of treatment. Similarly, hypothyroidism resulted in a 2.6-fold decrease in estrogen induction of galanin gene expression. The effect of thyroid hormone on anterior pituitary galanin gene expression appears to be exerted, at least in part, at the pituitary itself. Transient expression assays in GH3 cells suggest the involvement of transcriptional mechanisms in the regulation of galanin gene expression by thyroid hormone. A region between -41 and -132 bp upstream of the transcriptional start site confers thyroid hormone responsiveness to the galanin gene. Gel-mobility shift assays show specific binding of 'SPI-like' proteins in GH3 nuclear extracts to this region of the galanin gene. This binding was greatly enhanced by thyroid hormone.