Malignancy of pheochromocytomas is difficult to estimate on the basis of histopathological features. Good prognostic markers are not available. In our search for new markers to differentiate malignant pheochromocytomas from benign ones we tested the value of inhibin/activin subunit expression. Inhibins are heterodimeric glycoproteins consisting of an alpha-subunit and either a betaA- or a betaB-subunit. Activins are composed of beta-subunits only. Immunohistochemically inhibin/activin betaB-subunit was strongly positive in the normal adrenal medulla, but the cortex was negative. A striking difference was found in inhibin/activin betaB expression between benign and malignant pheochromocytomas. The majority of benign adrenal tumors (27 of 30) showed strong or moderate immunoreactivity, whereas all seven malignant tumors were negative or only weakly positive for inhibin/activin betaB-subunit. The percentage of positively staining cells varied greatly in extraadrenal pheochromocytomas and in those benign tumors that showed over 5 mitoses/10 high power fields, necrosis, or capsular or vascular invasion, here called borderline tumors. Inhibin/activin betaB messenger ribonucleic acid was also found in pheochromocytomas. However, no significant differences in messenger ribonucleic acid levels were found in various types of tumors. Weak immunohistochemical positivity for inhibin/activin betaA-subunit was detected in the adrenal cortex, but the medulla and most of the pheochromocytomas were negative. Our data show that inhibin/activin betaB-subunit is expressed in normal adrenal medullary cells. Strong staining is found in most benign adrenal pheochromocytomas, whereas malignant tumors are almost negative. This suggests that loss of inhibin/activin betaB-subunit expression in pheochromocytomas may be used as an indicator of malignant potential.