Nodular or multinodular goiter is the most common non-neoplastic thyroid disease and may be difficult to distinguish from true neoplastic thyroid diseases using microscopic criteria. We have used two-dimensional gel electrophoresis to study the protein patterns of thyroid tissues including normal thyroid, multinodular goiter, diffuse hyperplasia, follicular adenoma, follicular carcinoma and papillary carcinoma. Specific proteins, in the region of molecular mass 15-30 kDa and isoelectric point 4.5-6.5, were identified by electrospray tandem mass spectrometry and protein sequencing. The most distinctive protein found is cathepsin B, which could be detected as four spots, with differential expression in different thyroid diseases. In particular, two of these cathepsin B spots CB2 and CB3 are strongly up-regulated in neoplastic diseases, compared to non-neoplastic diseases. In addition, overexpression of ATP synthase D chain and prohibitin were observed in papillary carcinoma, which should allow it to be differentiated from follicular carcinoma. Changes in expression of other proteins were also observed in disease states compared to normal tissues, namely translationally controlled tumor protein, thioredoxin peroxidase 1, glutathione-S-transferase P, DJ-1 protein, superoxide dismutase (Cu, Zn), and heat shock protein 27, but these changes are less characteristic, so they do not allow the differentiation between neoplastic and non-neoplastic tissues. Thus, the proteomic approach is a useful diagnostic tool for studying diseases involving the thyroid nodule.