The adrenal gland is composed of the medulla and the cortex, which is further subdivided into three zones: zona glomerulosa (zG), zona fasciculata (zF) and zona reticularis (zR). The zones of the cortex are functionally characterised by their ability to synthesise different steroids and consequently they express different steroidogenic enzymes. These and other markers of the zones have been described but so far no good candidate for a determining factor of zonal establishment has been discovered. Bone morphogenetic proteins (BMPs) are multifunctional cytokines belonging to the transforming growth factor-β (TGF-β) superfamily. In a microarray analysis of transcripts from the rat adrenal zG and zF, we have discovered that some BMPs are potentially ZG specific and BMP-3B showed exclusive expression in zG by Real-Time PCR and immunohistochemistry. Adrenal H295R cells (human adrenocorticocarcinoma cell line) were used as an in-vitro model to examine the role of BMP-3B further. The cells were differentiated into a zG (by Angiotensin II) and zF (by Forskolin) phenotype in the presence and absence of exogenous BMP-3B protein. BMP-3B was able to drive the differentiation of H295R cells into a more zG phenotype while inhibiting the differentiation into a zF phenotype as judged by the inhibition of CYP11B1 expression and the promotion of CYP1B2 expression respectively. The effect of BMP-3B on differentiation was confirmed by over-expressing BMP-3B in stable cell lines and blocking endogenous BMP-3B by siRNA. These experiments imply a role for BMP-3B in steroidogenesis and by implication in adrenal zonation.