GH and IGF‐I are capable of inducing cellular hypertrophy and/or hyperplasia. Chronic overexpression of GH in transgenic mice results in systemically and locally increased IGF‐I levels and in disproportionate overgrowth, including adrenocortical enlargement and corticosterone hypersecretion. Using PEPCK‐bovine GH transgenic (G) mice, we demonstrate that adrenal enlargement involves both hypertrophy (44%) and hyperplasia (50%) of zona fasciculata cells. To clarify whether IGFBP‐2 affected cell volume and number, we crossed hemizygous G mice with hemizygous CMV‐IGFBP‐2 transgenic (B) mice, generating G mice, B mice, GB double transgenic mice, and nontransgenic controls (C). The absolute weight of the adrenal glands was significantly increased in 5‐wk‐and 4‐month‐old G mice vs. C and B mice. IGFBP‐2 overexpression in GB mice reduced this effect of GH excess by 26% and 37% in 5‐wk‐and 4‐month‐old animals, respectively. GH‐induced hypertrophy of zona fasciculata cells was completely abolished by IGFBP‐2 overexpression in GB mice whereas hyperplasia was not affected. Basal and ACTH‐induced plasma corticosterone levels of 4‐month‐old G mice, but not of GB mice, were two‐to threefold increased compared with C mice. Plasma ACTH levels were similar in all groups. Our data show that IGFBP‐2 potently separates hypertrophic and hyperplastic effects of GH/IGF‐I excess on adrenocortical cells.—Hoeflich, A., Weber, M. M., Fisch, T., Nedbal, S., Fottner, C., Elmlinger, M. W., Wanke, R., Wolf, E. Insulin‐like growth factor binding protein 2 (IGFBP‐2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF‐I excess on adrenocortical cells in vivo. FASEB J. 16, 1721–1731 (2002)