In an attempt to understand the mechanism behind the invasion and metastasis in adrenocortical cancer we performed mRNA in situ hybridization on 30 tumors for three matrix metalloproteinases (MMPs): gelatinase A, membrane type 1 matrix metalloproteinase (MT1-MMP), and collagenase-3. All are known to participate in the invasion and metastasis of other tumor forms by degrading the extracellular matrix. Thirteen of sixteen cancers, but only one of fourteen benign lesions showed expression of gelatinase A, which was localized in stromal cells. MT1-MMP is thought to assist in tumor invasion and metastasis by activating the zymogen gelatinase A. Of 14 malignant tumors analyzed, 12 showed MT1-MMP mRNA expression, which in 7 cases was detected in both neoplastic and stromal cells. The benign tumors showed MT1-MMP expression in only 3 of 11 cases, and it was restricted to tumor cells. Fourteen tumors (11 cancers, 3 adenomas) were also analyzed for collagenase-3 mRNA, but no expression was detected. In conclusion, our data show that gelatinase A mRNA is expressed in most malignant adrenocortical tumors but not in the benign tumors. Gelatinase A mRNA expression is restricted to stromal cells, whereas its activator, MT1-MMP, is expressed in both stromal and neoplastic cells. Inhibition of gelatinase A and other proteinases may in the future become important as a form of cancer treatment.