According to recent reports, substance P (SP) is localized in the anterior pituitary gland within subsets of thyrotropes and somatotropes, although earlier electron-microscopic studies described the presence of this tachykinin in mammotropes and gonadotropes. Transgenic mice overexpressing the growth-hormone-releasing hormone (GHRH) gene have markedly enlarged pituitary glands, due to hyperstimulation of the somatotropes. Therefore, we speculated that if somatotropes are able to synthesize tachykinins, these peptides should be greatly increased in the anterior pituitary of transgenic GHRH mice. We found that, in accordance with our working hypothesis, both SP and neurokinin A (NKA) were markedly increased in the anterior pituitary gland of male and female transgenic mice, compared with their respective normal controls. In male transgenic mice, NKA was 13.6- and SP 20.2-fold higher than in the anterior pituitary from normal mice. In female transgenic mice, NKA was 40- and SP 100-fold higher than in the anterior pituitary from normal female mice. In male transgenic mice, NKA and neuropeptide K (NPK) contents in the anterior pituitary showed no significant changes between 26 and 50 days of age but significantly increased between 50 days and 5 months of age. The concentration of NKA in the anterior pituitary did not show significant differences between 26 days and 5 months of age, but NPK concentrations in the anterior pituitary significantly decreased with age. In female transgenic mice, NKA content and concentration in the anterior pituitary increased after 35 days of age, but NPK concentrations significantly decreased after 26 days of age. Triiodothyronine markedly decreased anterior pituitary tachykinins, but ovariectomy and estrogen administration failed to significantly affect tachykinin concentrations in the anterior pituitary of transgenic mice. Tachykinin immunostaining was detected in some somatotropes, but tachykinins were also present in cells that were not GH positive. These findings indicate that hyperstimulated somatotropes contain increased stores of tachykinins and that these cells are a source of tachykinins in the anterior pituitary. Tachykinin stores in the anterior pituitary of transgenic mice were affected by thyroid hormones but seem to be insensitive to estrogens. The GHRH transgenic mice may be an interesting model to study the regulation of tachykinin stores in the anterior pituitary gland.