Intrinsic and extrinsic stimuli result in profound pituitary growth changes ranging from hypoplasia to hyperplasia. Pituitary tumor transforming gene (PTTG) abundance correlates with pituitary trophic status. Mice with Pttg inactivation exhibit pituitary hypoplasia while targeted pituitary PTTG over-expression driven by -subunit glycoprotein (GSU) promoter results in focal pituitary hyperplasia. To test the impact of pituitary hyperplasia on tumor development we cross-bred GSU.PTTG with Rb+/- mice, which develop pituitary tumors with high penetrance. Pituitary glands of resulting bi-transgenic GSU.PTTGxRb+/- mice were compared with mono-transgenic GSU.PTTG, Rb+/-, and wild-type mice. Confocal microscopy showed that PTTG over-expressing cells have enlarged nuclei and marked redistribution of chromatin, and electron microscopy of GSU.PTTG pituitaries showed enlarged gonadotrophs with prominent Golgi complex and numerous secretory granules. These morphological findings were even more remarkable in GSU.PTTGxRb+/- pituitaries. Mice from all 4 genotypes were sequentially imaged by magnetic resonance (MRI) to evaluate pituitary volume, and glands from GSU.PTTGxRb+/- mice were the largest as early as 2 months of age (P = 0.0003). Cumulative incidence of pituitary tumors visualized by MRI did not differ between Rb+/- and GSU.PTTGxRb+/- mice. However, anterior lobe tumors determined after necropsy were 3.5 times more frequent in GSU.PTTGxRb+/- than in Rb+/- mice (P = 0.0036), while the frequency of intermediate lobe tumors was similar. In summary, GSU.PTTGxRb+/- pituitary glands exhibit enhanced cellular activity, increased volume and higher prevalence of anterior pituitary tumors, indicating that changes in pituitary PTTG content directly relate to both pituitary trophic status and tumorigenic potential.