Cathepsin B (CB) and cathepsin L (CL) are cysteine endopeptidases involved in the processing of thyroglobulin (Tg) in the normal thyroid. As thyroglobulin expression is frequently altered in thyroid carcinomas, we have analyzed 42 human thyroid tissues from 40 patients to study the effect of malignant transformation an the expression of these endopeptidases. Our samples included 18 cases of papillary carcinoma (of which 10 also had matched adjacent normal thyroid tissue), 6 cases of normal thyroid from autopsy patients, 1 case of follicular carcinoma, 2 cases of medullary carcinoma, 2 cases of follicular adenoma, 3 cases of Hashimoto's thyroiditis (HT) and 10 samples from 8 patients with multi-nodular goiter (MNG). Enzyme-specific activities were increased 15-fold for CB and 9-fold for CL in papillary carcinoma compared with normal adjacent thyroid tissue or normal thyroid from autopsies. CB mRNA content was also markedly increased in papillary carcinoma compared with normal thyroid, primarily due to elevated levels of the 2.2-kb CB mRNA transcript. In thyroids with nonneoplastic diseases, including MNG and HT, there was no significant increase in either CB or CL enzyme activities nor CB mRNA levels compared with normal thyroids from non-cancer cases. Immunohistochemical studies on papillary carcinomas revealed increased CB staining in papillary carcinoma cells, with prominent staining close to the basement membranes of many of the neoplastic cells. Our observations suggest that CB and CL enzyme activities are potentially useful new biochemical markers for distinguishing papillary carcinoma of the thyroid from non-neoplastic thyroid disease.