The biological function of the inhibin-alpha subunit in gonadal tumorigenesis is different in human compared to mouse. The inhibin-alpha subunit is up-regulated in most human ovarian and testicular cancers but knock-out studies in mice showed the inhibin-alpha subunit is a tumour suppressor with gonadal and adrenal specificity. The inhibin-alpha subunit is a component of the inhibin/activin signalling pathway which includes activin receptors ActRIIA/IIB and intracellular Smads-2/3. To resolve the incongruity in function in human versus mouse, we re-evaluated the inhibin/activin pathway in human gonadal and adrenal cancers using contemporary protein and mRNA expression data for multiple pathway components rather than inhibin-alpha alone. We used an inhibin-alpha antibody raised against the N-terminal domain to compare immunoreactivity with the more commonly used antibody raised against the C-terminal domain. This study also described, for the first time, a comprehensive protein expression profile of activin-betaC in reproductive and adrenal cancers, and its effect on a human granulosa cell line, providing evidence for a role in ovarian, testis and adrenal tumour biology. Our data show reduced inhibin-alpha expression at both protein and mRNA levels, and increased activin signalling in human testicular, ovarian and malignant versus benign forms of adrenal cancer. We also found that activin-C acts as an activin-A antagonist by binding to activin receptor subunits IIA and IIB and modulating the canonical Smad pathway. In conclusion, analysis of the inhibin/activin signalling pathway helps to explain discrepancies arising from studies of only one hormone or subunit and suggests that altered expression of the inhibin and activin subunits is associated with reproductive and adrenal cancer biology.