The presence of activin A and its effects have previously been documented in the adrenal gland, particularly in the human fetal adrenal gland and the rat adrenal gland. The primary signaling pathway of activin involves interactions between receptor and intracellular (Smad) proteins that have not been completely described in the adrenal gland. In this study, we demonstrate that the components of the activin signaling cascade are present in two complementary models, the fetal rat adrenal gland and the human adrenocortical cell line, H295R, by means of RT-PCR, western analysis, and immunoprecipitation techniques. Using the cell line, activin signaling was analyzed using an activin-responsive reporter gene, p3TP-luc, and luciferase assays to assess transcriptional activity with co-expression of the different activin receptors and Smads to demonstrate the functionality of the signaling cascade. In the fetal rat adrenal gland, the relative amounts of mRNA of the type II receptors, RII and RIIB, were regulated by gestational age, such that the RIIB levels increased after birth while RII levels fell. Using immunodetection techniques, the activin receptors and the different Smad proteins were detected in the rat fetal adrenal glands. Notably, the presence of Smad4 protein is significantly increased after birth in the rat adrenal gland. RT-PCR established a similar profile in the H295R cells. Using p3TP-luc, the H295R cells show transcriptional activation of this activin-responsive reporter in the presence of activin A. Co-expression of type I and type II receptors as well as Smads, results in ligand-independent transcriptional activity in addition to an activin-stimulated response. In determining activin's effects on adrenal function, adrenal steroid production was evaluated by incubation of the H295R cells with increasing doses of activin A and inhibin A, resulting in a detectable increase in P450c17 expression. Co-incubation of activin A with follistatin diminishes this response. These results are consistent with a role for activin A in the adrenal gland by demonstrating that the elements of the activin signaling pathway are present, intact, and functional. This suggests that in the adrenal gland the components of the activin receptor/Smad pathway are dynamically changing in the transition from fetal to neonatal life, and are important to the function of this organ.