Previously we showed that thyroid-targeted overexpression of the PTTG binding factor (PBF) induced significant goitrogenesis in transgenic mice (PBF-Tg). In 10 week old PBF-Tg mice thyroid weight was increased by 1.8±0.3-fold compared to wild-type (WT) controls (n=20, P<0.0001). We have now examined the long-term effects of PBF overexpression on thyroid gland weight and histology in aged PBF-Tg mice. Our study shows that the penetrance of increased thyroid weight was 100% with all PBF-Tg mice demonstrating markedly enlarged thyroid glands (n=256). By 12 months of age PBF-Tg thyroid glands were 3.2-fold larger than that of WT littermates (n=22, P<0.0001), which persisted to 18 months of age (2.7±0.5-fold, n=27, P<0.0001). Histological examination showed that PBF-Tg mice were prone to macrofollicular lesions with >90% of mice (n=11/12) demonstrating follicles >250 microns in diameter by 18 months of age (P=0.02 compared with WT). Focal and nodular hyperplasia was also apparent, with 75% of PBF-Tg mice (n=9/12) demonstrating evidence of hyperplastic lesions by 18 months of age (P=0.009 compared with WT). Western blot analysis showed a significant 3.4±1.2-fold (n=7, P=0.0007) activation of phosphorylated Akt, a known regulator of thyroid cell proliferation, in transgenic thyroids. In contrast there was no significant increase in total Akt levels. Positive immunostaining with the proliferation marker cyclin D1 in diffuse goitre regions was significantly higher in aged PBF-Tg mice (n=4834/35958 positive cells) compared to WT mice (n=1231/25987 positive cells; P<0.0001). Further, hyperplastic lesions of aged PBF-Tg mice demonstrated much greater cyclin D1 staining, ranging from 19 to 68% of cells, with a mean value of 37.7±20.7% (n=5). These results demonstrate that targeted overexpression of PBF induces goitre in vivo, and causes significant hyperplastic growth of the thyroid gland. Further, our findings implicate the Akt pathway in mediating PBF-induced thyroid cell proliferation in vivo.