The differential diagnosis of adrenocortical carcinoma from adrenocortical adenoma is based on different pathological parameters, usually incorporated in scoring systems, which unfortunately lack a 100% sensitivity and specificity. Little is known on the molecular mechanisms leading to the malignant phenotype in adrenocortical tumors. Among other molecules, metalloproteinases were demonstrated to be implicated in malignant progression and metastatization of solid tumors, including endocrine ones. Therefore, we aimed to investigate metalloproteinases and their inhibitors expression in a series of 50 adrenocortical carcinomas and 50 control adrenocortical adenomas, diagnosed according to the Weiss histological criteria. Immunohistochemical results were scored by semiquantitative analysis and compared with clinicopathological parameters and outcome. Metalloproteinase type 2 gave the most significant result, being detected in neoplastic cells in 1/50 adrenocortical adenomas (2%) and 37/50 adrenocortical carcinomas (74%) (P < 0.001), with a focal (score 1, <20% of positive cells--two-thirds of cases) or diffuse (score 2, >20% of positive cells--one-third of cases) pattern. In addition, diffuse (score 2) metalloproteinase type 2 protein expression, as compared to focal or negative immunostaining, correlated with shorter survival (P < 0.02) and disease-free interval (P = 0.05). No correlation was found comparing metalloproteinase type 2 expression and any clinicopathological parameter. Our data indicate that metalloproteinase type 2 immunohistochemical localization in tumor cells is significantly restricted to malignant adrenocortical tumors, with high specificity but low sensitivity. In addition, a strong metalloproteinase type 2 expression in adrenocortical carcinoma was for the first time recognized as an unfavorable prognostic factor.